Solubilization of antigen components for removal from tissues

ABSTRACT

The present invention relates to methods for removing antigens from tissues by sequentially destabilizing and/or depolymerizing cytoskeletal components and removing and/or reducing water-soluble antigens and lipid-soluble antigens. The invention further relates to tissue scaffolding and decellularized extracellular matrix produced by such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/612,964 filed on Mar. 19, 2012 and U.S.Provisional Application No. 61/727,738 filed on Nov. 18, 2012, both ofwhich are hereby incorporated herein by reference in their entirety forall purposes.

FIELD

Provided are methods for removing antigens from tissues by sequentiallyremoving and/or reducing water-soluble antigens and lipid-solubleantigens. Also provided are tissue scaffolding and decellularizedextracellular matrix produced by such methods.

Further provided are methods for removing antigens from tissues bysequentially destabilizing and/or depolymerizing cytoskeletal componentsand removing and/or reducing water-soluble antigens and lipid-solubleantigens. Also provided are tissue scaffolding and decellularizedextracellular matrix produced by such methods.

BACKGROUND

The ability of xenoantigens to elicit the immune response represents thecritical barrier in the generation of scaffolds from xenogeneic tissuesfor tissue engineering and regenerative medicine applications (Platt, etal., Circulation. (2002) 106:1043-1047). Decellularization approacheswere originally developed with the intention of addressing antigens inxenogeneic tissues. The decellularization paradigm attributes xenograftantigenicity to the cellular component of a tissue and uses the absenceof cells by light microscopy as the principle determinant of success ofthe process. Implantation of decellularized porcine valve tissue intosheep, rats, and dogs showed little immunogenic response for up to oneyear, encouraging confidence in decellularization methods (Goldstein, etal., Ann. Thorac. Surg. (2000) 70:1962-1969; Iwai, et al., J. ArtificialOrgans. (2007) 10:29-35). Unfortunately, in vivo studies have reportedthe rapid failure of SynerGraft decellularized porcine heart valvesfollowing implantation into juvenile patients (Simon, et al, Eur. J.Cardiothorac. Surg. (2003) 23:1002-1006). Failure of the acellularSynerGraft prosthetic has been attributed to inadequate xenoantigenremoval with decellularization (Simon, et al., supra). Foreign body typereaction and inflammatory cell infiltration into implanted SynerGraftvalves has also been demonstrated (Simon, et al., supra; Sayk, et al.,Ann. Thorac. Surg. (2005) 79:1755-1758). Additionally, persistentcellular debris following the SynerGraft decellularization process hasbeen shown to be sufficient to elicit an immunogenic reaction andcalcification (Schmidt, et al., Biomaterials. (2000) 21:2215-2231).Recent studies have indicated that acellularity on light microscopy doesnot equate to removal of known xenoantigens from the biomaterial(Goncalves, et al., J. Heart Valve Dis. (2005) 14:212-217; Meyer, etal., J. Biomed. Mater. Res. A. (2006) 79A:254-262; Wong, et al., ActaBiomaterialia (2013) epub ahead of print (doi:10.1016/j.actbio.2012.12.034). We have shown the lack of correlationbetween residual nuclei counts in bovine pericardium (BP) and residualwater-soluble protein (WSP) antigenicity of the biomaterial (Wong, etal., Biomaterials (2011) 32:8129-8138). Taken together, these resultsindicate that the fundamental principles behind the use ofdecellularization as the sole process necessary for xenogeneic scaffoldgeneration and principal determinant of biomaterial antigenicity appearto be flawed. Thus, a void remains in the development of an antigenremoval (AR) process to effectively reduce xenogeneic scaffoldantigenicity.

A critical error in previous decellularization approaches was focusingmerely on cell disruption without regard to the need for the antigenicmolecules to be solubilized for efficient removal from the xenogeneictissue. We have demonstrated previously that by promoting thesolubilization of WSPs using a reducing agent and salt to preventintermolecular aggregation and subsequent precipitation from solution,removal of WSP antigens from BP is significantly enhanced (Wong, et al.,Biomaterials, supra). Our solubilization-based AR approach reduced theresidual WSP antigenicity of BP by an additional 80% compared tohypotonic solution and 60% compared to 0.1% (w/v) sodium dodecyl sulfate(SDS) decellularization methods while maintaining biomaterial tensileproperties and extracellular matrix (ECM) structure and composition(Wong, et al., Biomaterials, supra). However, by only promoting WSPsolubilization for removal, lipid-soluble protein (LSP) antigens arelikely to persist within the tissue. Thus, a means to encourage LSPsolubilization for subsequent removal, following initial WSPsolubilization, could reduce overall residual antigenicity in BP post-AR(BP-AR).

The concept of differential protein solubility has long been recognizedin proteomics wherein a sequential, differential approach is used forthe serial extraction of protein fractions from a homogenized tissue.Protein extraction methods exploit the physiochemical properties ofproteins in order to differentially and sequentially extract varioussubsets of proteins for downstream analyses (Beers, et al., Am. J.Physiol. (1992) 262:L773-778; DuPont. J. Agric. Food Chem. (2005)53:1575-1584; Cordwell, et al., Methods Mol. Biol. (2008) 424:139-146;Wilson, et al., Matrix Biol. (2008) 27:709-712). The use of a series ofsolutions to promote protein solubilization along a spectrum ofsolubilities (e.g., WSP extraction followed by LSP extraction) iscritical for such sequential, differential extraction protocols, sinceproteins can only be extracted from the material into solutions in whichthey are soluble (Cordwell, et al., Methods Mol. Biol., supra). However,the importance of promoting sequential, differential protein solubilityduring AR from intact tissues in the generation of xenogeneic scaffoldshas not been investigated. This is a surprising oversight given thecomplex composition of protein antigens within a tissue requiringremoval prior to implementation in tissue engineering applications.

The present invention is based, in part, on the discovery that a seriesof solutions, each promoting the solubilization and subsequent removalof a different subset of tissue proteins based on their solubility,enhances overall AR from BP. Furthermore, such a sequential,differential AR strategy significantly reduces BP antigenicity whilemaintaining biomaterial functional properties. In this study, severalLSP solubilization promoting agents were applied as a second step of ARfollowing initial WSP solubilization and assessed for their ability toreduce the residual LSP antigenicity of the resultant BP-AR. Theeffectiveness of this two-step sequential, differential strategy forreducing LSP antigens in BP was compared to a one-step AR strategy (WSPsolubilization) (Wong, et al., Biomaterials, supra) and the literaturegold standard (1% (w/v) SDS). See, Wong, et al., Acta Biomaterialia(2013) epub ahead of print (doi: 10.1016/j.actbio.2012.12.034). Theeffect of this two-step AR protocol on ECM mechanical properties,structure and composition of BP-AR was assessed by uniaxial tensiletesting, histological analysis and biochemical quantification of ECMcomponents, respectively.

Previously described myocardial tissue decellularization methods havealso been solely based on the ubiquitous use of harsh denaturingdetergents, mainly SDS, in concentrations as high as 2% in hypotonicwater solutions. See, Elder, et al., Biomaterials. (2009) 30(22):3749-3756. Although effective in solubilizing cellular and tissuecomponents, it has been shown that these methods are not successful inremoving antigenic determinants, while they are often detrimental to theextracellular matrix (removing elastin, glycosaminoglycans and damagingcollagen structure). Further, commonly utilized detergents are toxic torepopulating cells reducing the chances of successful recellularizationstrategies for the produced scaffolds.

Reported methods to produce a myocardial scaffold have been based ondetergent-based decellularization methods. More specifically, theseapproaches included the use of one detergent (SDS, Triton-X100, Saponin)with protease inhibitors (to prevent extracellular matrix proteindegradation) and occasionally enzymatic treatments (such as trypsin) andnucleases for nucleic acid degradation in different concentrations andcombinations. All these reports determine loss of nuclei and cellularcomponents and production of an acellular scaffold as their outcomemeasure for protocol success. As we have already shown in our laboratorythis assumption is not valid, since antigens may be associated withnon-cellular components of the tissue. Additionally, the omission of areducing agent in all these treatments would be expected to result inprotein precipitation rather than solubilization and extraction,regardless of the concentration of the detergent used.

SUMMARY

The present invention relates to methods for removing antigen componentsfrom tissue, e.g., to create a tissue scaffolding and/or a substantiallydecellularized extracellular matrix, e.g., for use in tissuetransplantation, tissue regeneration, and/or model matrices for study ofcellular/ECM interactions. In various embodiments, the tissue is anintact tissue. As appropriate, the tissue may be a part of an organ oran intact organ. Generally, the methods are performed in vitro.

In various embodiments, the methods comprise sequentially solubilizingand removing water soluble antigen components and solubilizing andremoving lipid-soluble antigen components. In various embodiments, thewater-soluble antigen components are first solubilized and removed fromthe tissue, and then the lipid-soluble antigen components aresubsequently solubilized and removed from the tissue. In variousembodiments, the lipid-soluble antigen components are first solubilizedand removed from the tissue, and then the water-soluble antigencomponents are subsequently solubilized and removed from the tissue.

In various embodiments, the methods comprise sequentially, destabilizingand/or depolymerizing cytoskeletal components (e.g., filamentous actinand/or microtubules) to solubilize the macromolecular structure of thecellular cytoskeletion and thereby facilitate solubilization and removalof those water-soluble antigen components and lipid-soluble antigencomponents which are associated with the cytoskeleton In variousembodiments, the tissue is first contacted with a solution comprisingone or more cytoskeletal destabilizing and/or depolymerizing agents. Invarious embodiments, the water-soluble antigen components aresolubilized and removed from the tissue, and then the lipid-solubleantigen components are subsequently solubilized and removed from thetissue. In various embodiments, the lipid-soluble antigen components aresolubilized and removed from the tissue, and then the water-solubleantigen components are subsequently solubilized and removed from thetissue.

Accordingly, in one aspect, the invention provides methods of removingimmunogenic antigens from a tissue. In some embodiments, the methodscomprise:

-   -   a) solubilizing water-soluble antigens in the tissue;    -   b) separating the tissue from the solubilized water-soluble        antigens;    -   c) solubilizing lipid-soluble antigens in the tissue; and    -   d) separating the tissue from the solubilized lipid-soluble        antigens; thereby removing immunogenic antigens from the tissue.

In one aspect, the invention provides methods for removing immunogenicantigens from a tissue. In some embodiments, the methods comprise:

-   -   a) contacting the tissue with one or more cytoskeletal        destabilizing and/or depolymerizing agents;    -   b) solubilizing water-soluble antigens in the tissue;    -   c) separating the tissue from the solubilized water-soluble        antigens;    -   d) solubilizing lipid-soluble antigens in the tissue; and    -   e) separating the tissue from the solubilized lipid-soluble        antigens; thereby removing immunogenic antigens from the tissue.        In various embodiments, a wash or rinse step is performed after        step a), separating the tissue from the one or more cytoskeletal        destabilizing agents and destabilized and/or depolymerized        cytoskeletal proteins and associated antigens. In varying        embodiments, the steps of the method are performed in the order        set forth above. In varying embodiments, one or more of the        steps are repeated, e.g., one, two, three or more times, as        appropriate or desired.

In a related aspect, the invention provides methods for removingimmunogenic antigens from a muscle tissue, comprising:

-   -   a) relaxing the muscle tissue in a relaxing solution comprising        an energy source molecule or other molecule to dissociate        actin-myosin crossbridges;    -   b) contacting the muscle tissue with one or more cytoskeletal        destabilizing and/or depolymerizing agents;    -   c) contacting the muscle tissue with a concentrated salt        solution;    -   d) solubilizing water-soluble antigens in the tissue;    -   e) separating the muscle tissue from the solubilized        water-soluble antigens;    -   f) solubilizing lipid-soluble antigens in the tissue; and    -   g) separating the muscle tissue from the solubilized        lipid-soluble antigens; thereby removing immunogenic antigens        from the muscle tissue. In various embodiments, a wash or rinse        step is performed after step a), separating the tissue from the        relaxing solution. In various embodiments, a wash or rinse step        is performed after step b), separating the tissue from the one        or more cytoskeletal destabilizing agents and destabilized        and/or depolymerized cytoskeletal proteins. In various        embodiments, a wash or rinse step is performed after step c),        separating the tissue from the concentrated salt solution and        solubilized sarcomeric components. In varying embodiments, the        steps of the method are performed in the order set forth above.        In varying embodiments, the steps for removing immunogenic        antigens from a muscle tissue are performed in the following        order: a), b), c), d), e), f) and g). In varying embodiments,        the steps for removing immunogenic antigens from a muscle tissue        are performed in the following order: a), f), g), b), c), d) and        e). In varying embodiments, the steps for removing immunogenic        antigens from a muscle tissue are performed in the following        order: a), d), e), b), c), f) and g). In varying embodiments,        the steps for removing immunogenic antigens from a muscle tissue        are performed in the following order: a), d), e), f), g), b) and        c). In varying embodiments, one or more of the steps are        repeated, e.g., one, two, three or more times, as appropriate or        desired. For example, in some embodiments, steps b), c), d)        and e) are repeated one or more times. In varying embodiments,        the steps for removing immunogenic antigens from a muscle tissue        are performed in the following order: a), b), c), d), e), f),        g), b), c), and e). In varying embodiments, the steps for        removing immunogenic antigens from a muscle tissue are performed        in the following order: a), f), g), b), c), d), e), f), and g).

In varying embodiments of the methods, the lipid-soluble antigens aresolubilized and separated first, and then the water-soluble antigens aresolubilized and separated. In varying embodiments, the water-solubleantigens are solubilized and separated first, and then the lipid-solubleantigens are solubilized and separated. In varying embodiments, thetissue is contacted with one or more one or more cytoskeletaldestabilizing and/or depolymerizing agents concurrently with thesolubilization and separation of water soluble antigens.

With respect to further embodiments of the methods, in some embodiments,the immunogenic antigens are selected from the group consisting ofprotein antigens, lipid antigens and carbohydrate antigens. In someembodiments, the method does not comprise contacting the tissue with adetergent selected from the group consisting of sodium dodecyl sulfate,Triton-X-100, Triton-X-114, Triton-X-200 and sodium deoxycholate. Insome embodiments, the method does not comprise contacting the tissuewith a protease, for example, trypsin.

In some embodiments, the one or more cytoskeletal destabilizing agentscomprise one or more actin depolymerization agents. In some embodiments,the one or more actin depolymerization agents are selected from thegroup consisting of Cytochalasin A, Cytochalasin B, Cytochalasin C,Cytochalasin D, Cytochalasin E, Cytochalasin F, Cytochalasin G,Cytochalasin H, Cytochalasin I, Cytochalasin J, Latrunculin A,Latrunculin B, Swinholide A, Misakinolide A, Bistheonelide A,Scytophycin A, Scytophycin B, Scytophycin D, Scytophycin E,19-O-Demethylscytophycin C, 6-Hydroxyscytophycin B,6-Hydroxy-7-o-methylscytophycin E and tolytoxin, Mycalolide A,Mycalolide B, Mycalolide C, secomycalolide A and 30-hydroxymycalolide A,Halichondramide, (19Z)-halichondramide, kabiramides B, kabiramides C,kabiramides D, kabiramides G, kabiramides J, kabiramides K, ulapualideA, jaspamide, Dihydrohalichondramide, Aplyronine A, Aplyronine B,Aplyronine C, Pectenotoxin 2, Pectenotoxin 6, and Migrastatin. In someembodiments, the actin depolymerization agents depolymerize filamentouscytoskeletal actin (F-actin). In some embodiments, the actindepolymerization agents depolymerize filamentous α-sarcomeric actin(F-actin).

In some embodiments, the one or more cytoskeletal destabilizing agentscomprise one or more microtubule depolymerization or destabilizingagents. In some embodiments, the one or more microtubuledepolymerization or destabilizing agents is selected from the groupconsisting of colchicine, colcemid, vinblastine, vincristine,myoseverin, nocodazole, podophyllotoxin, polygamain and taxol.

In some embodiments, the water-soluble antigens are solubilized in asolution comprising a buffering agent, a reducing agent, a proteaseinhibitor, and one or more salts suitable for maintaining proteinsolubility. In some embodiments, the buffering agent maintains a pH inthe range of about 4-11, e.g., a pH in the range of about 4-6, 8-11,5-10 or 6-9. In some embodiments, the buffering agent maintains a pH ofat least about 8. In some embodiments, the one or more salts comprise amonovalent or a divalent anion. In some embodiments, the one or moresalts comprise a metal halide salt. In some embodiments, the metalhalide salt is selected from the group consisting of LiF, LiCl, LiBr,LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, RbF, RbCl, RbBr, RbI, CsF,CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂, BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂,CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂, SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂,BaI₂, and mixtures thereof. In some embodiments, the reducing agent isselected from the group consisting of Tributylphosphine (TBP), betamercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine(TCEP), cysteine-HCl, and dithiothreitol (DTT). In some embodiments, thewater-soluble antigens are solubilized in a solution that comprises oneor more of an antibacterial agent and/or an antifungal agent. In someembodiments, the water-soluble antigens are solubilized in a solutionthat comprises a chelation agent. In some embodiments, the chelationagent is selected from the group consisting ofethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), Citric Acid, N,N-bis(carboxymethyl)glycine (NTA), and themeso isomer of Dimercaptosuccinic acid (DMSA). In some embodiments, thewater-soluble antigens are solubilized in a solution that does notcomprise an amphiphile. In some embodiments, the water-soluble antigensare solubilized in a solution that does not comprise a detergent. Insome embodiments, the water-soluble antigens are solubilized in asolution that comprises a non-detergent sulfobetaine. In someembodiments, the water-soluble antigens are solubilized in a solutioncomprising Tris-HCl, dithiothreitol (DTT), a protease inhibitor, andKCl.

In some embodiments, the lipid-soluble antigens are solubilized in asolution comprising a buffering agent, a reducing agent, a proteaseinhibitor, one or more salts suitable for maintaining protein solubilityand an amphiphile. In some embodiments, the amphiphile is a zwitterionicdetergent. In some embodiments, the amphiphile is a sulfobetaine. Insome embodiments, the sulfobetaine is selected from the group consistingof 3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(amidosulfobetaine-14; ASB-14); amidosulfobetaine-16 (ASB-16);4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine (ASB-C8Ø);3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB 3-8);N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10),N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-12),N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-14);3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16);3-(N,N-Dimethyloctadecylammonio) propanesulfonate (SB3-18);3-(1-Pyridinio)-1-propanesulfonate (NDSB-201); 3-(Benzyldimethylammonio)propanesulfonate (NDSB-256); NDSB-211, NDSB-195, NDSB-221;3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), and mixtures thereof. In some embodiments, the lipid-solubleantigens are solubilized in a solution comprising Tris-HCl,dithiothreitol (DTT), a protease inhibitor, KCl and ASB-14.

In some embodiments, the method yields a substantially intactextracellular matrix (ECM) compatible with viable cell repopulation. Insome embodiments, the methods further comprise repopulating the ECM withlive cells. In some embodiments, the live cells are autologous,allogeneic or xenogeneic to the ECM. In some embodiments, the live cellscomprise mesenchymal stem cells. In some embodiments, the live cellscomprise cells of the same tissue type as the tissue from which theantigens are removed.

In some embodiments, the tissue is epithelial tissue, endothelialtissue, muscle tissue, or connective tissue. In some embodiments, thetissue is selected from the group consisting of cardiac muscle tissue,striated or skeletal muscle tissue, or smooth muscle tissue, heart,pericardium, heart valve, vessel, vascular conduit, artery, vein, skin,dermis, pericardium, dura, intestinal submucosa, ligament, tendon, bone,cartilage, ureter, urinary bladder, kidney, skin, lung, liver, andumbilical cord. In some embodiments, the tissue is an intact tissue. Insome embodiments, the tissue is within or a part of an intact organ. Insome embodiments, at least about 80%, for example, at least about 85%,90%, 93%, 95%, 97%, 99%, or more, of the water soluble antigens areremoved from the tissue. In some embodiments, at least about 60%, forexample, at least about 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%,99%, or more, of the lipid soluble antigens are removed from the tissue.

In some embodiments, the tissue is muscle tissue. In some embodiments,the muscle tissue is cardiac muscle tissue, striated or skeletal muscletissue, or smooth muscle tissue. In some embodiments, the methodcomprises the step of relaxing the muscle tissue prior to contacting thetissue with one or more cytoskeletal destabilizing agents. In someembodiments, the muscle tissue is relaxed in a relaxing solutioncomprising an energy source molecule or other actin-myosin dissociationagent. In some embodiments, the energy source molecule is selected fromthe group consisting of a nucleotide 5′-triphosphate (NTP), adenosine,inosine, aspartate, glutamate, creatine phosphate, a Kreb's cycleprecursor or intermediate, glucose, and dextrose. In some embodiments,the energy source molecule is pyrophosphate (PPi) or a nucleotide5′-triphosphate (NTP) selected from the group consisting of adenosine5′-triphosphate (ATP), inosine 5′-triphosphate (ITP), guanidine5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP), and uridine5′-triphosphate (UTP). In some embodiments, the energy source moleculeis a precursor of adenosine 5′-triphosphate (ATP). In some embodiments,the energy source molecule is adenosine 5′-triphosphate (ATP). In someembodiments, the energy source molecule is Pyrophosphate (PPi). In someembodiments, the energy source molecule comprises vanadate and adenosine5′-diphosphate (ADP). In some embodiments, the relaxing solution furthercomprises a calcium ion chelating agent. In some embodiments, therelaxing solution further comprises a permeabilization agent. In someembodiments, the muscle tissue is contacted with a concentrated saltsolution. In some embodiments, the concentrated salt solution comprisesone or more salts in a concentration range from about 0.5 M to about 3.0M, e.g., about 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M,1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M,2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, or 3.0 M. In some embodiments,the concentrated salt solution comprises one or more metal halide salts.In some embodiments, the metal halide salt is selected from the groupconsisting of LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr,KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂,BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂,SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂, BaI₂, and mixtures thereof. In someembodiments, the concentrated salt solution comprises KCl and KI. Insome embodiments, the concentrated salt solution comprises 0.6 M KCl and1.0 M KI. In some embodiments, sarcomeric constituents are notdetectable in the muscle tissue. In some embodiments, at least about 90%of the sarcomeric constituents are removed, e.g., at least about 93%,95%, 97%, 98%, 99% or all (100%) sarcomeric constituents are removed.

In a further aspect, the invention provides tissue scaffolds produced bythe methods described above and herein. In another aspect, the inventionprovides kits comprising a tissue scaffold produced by the methodsdescribed above and herein. In a related aspect, the invention providesdecellularized extracellular matrix (ECM) produced by the methodsdescribed above and herein. In varying embodiments, the ECM and/ortissue scaffolds are free of residual sodium dodecyl sulfate. In varyingembodiments, the ECM and/or tissue scaffolds induce little or no immuneresponse in a host that is xenogeneic or allogeneic to the ECM. In someembodiments, the ECM and/or tissue scaffolds do not comprise detectablesarcomeric consituents. In varying embodiments, the ECM comprises ECMstructure, ECM biochemical composition and ECM tensile strength that issubstantially the same as the ECM prior to decellularization, whereinthe ECM is compatible with viable cell repopulation, and issubstantially free of endogenous antigens.

In another aspect, the invention provides kits comprising (i) a solutionfor solubilizing water-soluble antigens and (ii) a solution forsolubilizing lipid-soluble antigens. In a further aspect, the inventionprovides kits comprising (i) a solution for destabilizing cytoskeletalpolymers, (ii) a solution for solubilizing water soluble antigens and(iii) a solution for solubilizing lipid soluble antigens. Furtherembodiments, of the solution for solubilizing water-soluble antigens andof the solution for solubilizing lipid-soluble antigens are as describedherein. In some embodiments, the kits further comprise a control tissuescaffold and/or a decellularized extracellular matrix (ECM) produced bythe methods described herein.

With respect to embodiments of the kits, in some embodiments, thesolution for destabilizing cytoskeletal polymers comprises one or moreactin depolymerization agents. In some embodiments, the one or moreactin depolymerization agents are selected from the group consisting ofCytochalasin A, Cytochalasin B, Cytochalasin C, Cytochalasin D,Cytochalasin E, Cytochalasin F, Cytochalasin G, Cytochalasin H,Cytochalasin I, Cytochalasin J, Latrunculin A, Latrunculin B, SwinholideA, Misakinolide A, Bistheonelide A, Scytophycin A, Scytophycin B,Scytophycin D, Scytophycin E, 19-O-Demethylscytophycin C,6-Hydroxyscytophycin B, 6-Hydroxy-7-o-methylscytophycin E and tolytoxin,Mycalolide A, Mycalolide B, Mycalolide C, secomycalolide A and30-hydroxymycalolide A, Halichondramide, (19Z)-halichondramide,kabiramides B, kabiramides C, kabiramides D, kabiramides G, kabiramidesJ, kabiramides K, ulapualide A, jaspamide, Dihydrohalichondramide,Aplyronine A, Aplyronine B, Aplyronine C, Pectenotoxin 2, Pectenotoxin6, and Migrastatin. In some embodiments, the actin depolymerizationagents depolymerize filamentous cytoskeletal actin. In some embodiments,the actin depolymerization agents depolymerize α-sarcomeric actin(F-actin). In some embodiments, the solution for destabilizingcytoskeletal polymers comprises one or more microtubule depolymerizationor destabilizing agents. In some embodiments, the one or moremicrotubule depolymerization or destabilizing agents is selected fromthe group consisting of colchicine, colcemid, vinblastine, vincristine,myoseverin, nocodazole, podophyllotoxin, polygamain and taxol.

In some embodiments, the solution for solubilizing water solubleantigens comprises a buffering agent, a reducing agent, a proteaseinhibitor, and one or more salts suitable for maintaining proteinsolubility. In some embodiments, the buffering agent maintains a pH inthe range of about 4-11, e.g., a pH in the range of about 4-6, 8-11,5-10 or 6-9. In some embodiments, the buffering agent maintains a pH ofat least about 8. In some embodiments, the one or more salts comprise amonovalent or a divalent anion. In some embodiments, the one or moresalts comprise a metal halide salt. In some embodiments, the metalhalide salt is selected from the group consisting of LiF, LiCl, LiBr,LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr, KI, RbF, RbCl, RbBr, RbI, CsF,CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂, BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂,CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂, SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂,BaI₂, and mixtures thereof. In some embodiments, the reducing agent isselected from the group consisting of Tributylphosphine (TBP), betamercaptoethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine(TCEP), cysteine-HCl, and dithiothreitol (DTT). In some embodiments, thesolution for solubilizing water soluble antigens comprises one or moreof an antibacterial agent and/or an antifungal agent. In someembodiments, the solution for solubilizing water soluble antigenscomprises a chelation agent. In some embodiments, the chelation agent isselected from the group consisting of ethylenediaminetetraacetic acid(EDTA), diethylenetriaminepentaacetic acid (DTPA), Citric Acid,N,N-bis(carboxymethyl)glycine (NTA), and the meso isomer ofDimercaptosuccinic acid (DMSA). In some embodiments, the solution forsolubilizing water soluble antigens does not comprise an amphiphile. Insome embodiments, the solution for solubilizing water soluble antigensdoes not comprise a detergent. In some embodiments, the solution forsolubilizing water soluble antigens comprises a non-detergentsulfobetaine. In some embodiments, the solution for solubilizing watersoluble antigens comprises Tris-HCl, dithiothreitol (DTT), a proteaseinhibitor, and KCl.

In some embodiments, the solution for solubilizing lipid solubleantigens comprises a buffering agent for maintaining pH of at least 8.0,a reducing agent, a protease inhibitor, one or more salts suitable formaintaining protein solubility and an amphiphile. In some embodiments,the amphiphile is a zwitterionic detergent. In some embodiments, theamphiphile is a sulfobetaine. In some embodiments, the sulfobetaine isselected from the group consisting of3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(amidosulfobetaine-14; ASB-14); amidosulfobetaine-16 (ASB-16);4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine (ASB-C8Ø);3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB3-8);N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10),N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-12),N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-14);3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16);3-(N,N-Dimethyloctadecylammonio) propanesulfonate (SB3-18);3-(1-Pyridinio)-1-propanesulfonate (NDSB-201); 3-(Benzyldimethylammonio)propanesulfonate (NDSB-256); NDSB-211, NDSB-195, NDSB-221;3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), and mixtures thereof. In some embodiments, the lipid-solubleantigens are solubilized in a solution comprising Tris-HCl,dithiothreitol (DTT), a protease inhibitor, KCl and ASB-14.

In some embodiments, the kits further comprise (iv) a relaxing solutioncomprising an energy source molecule. In some embodiments, the energysource molecule is selected from the group consisting of a nucleotide5′-triphosphate (NTP), adenosine, inosine, aspartate, glutamate,creatine phosphate, a Kreb's cycle precursor or intermediate, glucose,and dextrose. In some embodiments, the energy source molecule ispyrophosphate (PPi) or a nucleotide 5′-triphosphate (NTP) selected fromthe group consisting of adenosine 5′-triphosphate (ATP), inosine5′-triphosphate (ITP), guanidine 5′-triphosphate (GTP), cytidine5′-triphosphate (CTP), and uridine 5′-triphosphate (UTP). In someembodiments, the energy source molecule is a precursor of adenosine5′-triphosphate (ATP). In some embodiments, the energy source moleculeis adenosine 5′-triphosphate (ATP). In some embodiments, the energysource molecule is Pyrophosphate (PPi). In some embodiments, the energysource molecule comprises vanadate and adenosine 5′-diphosphate (ADP).In some embodiments, the relaxing solution further comprises a calciumion chelating agent. In some embodiments, the energy source molecule isa precursor of adenosine 5′-triphosphate (ATP). In some embodiments, therelaxing solution further comprises a permeabilization agent.

In some embodiments, the kits further comprise (v) a concentrated saltsolution. In some embodiments, the concentrated salt solution comprisesone or more salts in a concentration range from about 0.5 M to about 3.0M, e.g., about 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M,1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M,2.4 M, 2.5 M, 2.6 M, 2.7 M, 2.8 M, 2.9 M, or 3.0 M. In some embodiments,the concentrated salt solution comprises one or more metal halide salts.In some embodiments, the metal halide salt is selected from the groupconsisting of LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr,KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂,BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂,SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂, BaI₂, and mixtures thereof. In someembodiments, the concentrated salt solution comprises KCl and KI. Insome embodiments, the concentrated salt solution comprises 0.6 M KCl and1.0 M KI.

DEFINITIONS

The terms “tissue” or “biological tissue” interchangeably refer to acollection of interconnected cells and extracellular matrix that performa similar function or functions within an organism. Biological tissuesinclude, without limitation, connective tissue, muscle tissue, nervoustissue (of the brain, spinal cord, and nerves), epithelial tissue, andorgan tissue. Connective tissue includes fibrous tissue, e.g., fascia,tendon, ligaments, heart valves, bone, and cartilage. Muscle tissueincludes skeletal muscle tissue, smooth muscle tissue, e.g., esophageal,stomach, intestinal, bronchial, uterine, urethral, bladder, and bloodvessel tissue, and cardiac muscle tissue. Epithelial tissue includessimple epithelial tissue, e.g., alveolar epithelial tissue, blood vesselendothelial tissue, and heart mesothelial tissue, and stratifiedepithelial tissue. The biological tissue can additionally be selected,without limitation, from the group consisting of heart valve, vessel,vascular conduit, artery, vein, skin, dermis, pericardium, dura,intestinal submucosa, ligament, tendon, bone, cartilage, ureter, urinarybladder, liver, lung, umbilical cord, and heart. Multiple tissues/tissuetypes comprise organs. Organs are included herein under the terms“tissue and/or “biological tissue.”

The phase “intact tissue” refers to tissue that has not been minced orhomogenized. The intact tissue may be a whole tissue or a completeorgan.

The term “organ” as used herein refers to a collection of tissues joinedin a structural unit to serve a common function.

The term “dermis” as used herein refers to the layer of skin between theepidermis and the subcutaneous tissues.

The term “epithelial tissue” as used herein refers to the tissuecovering the whole surface of the body or lining certain organ systemsexposed to the external environment, such as the gastrointestinal tract,the urogenital tract, or the lung. It is made up of cells closely packedand arranged in at least one layer. This tissue is specialized to form acovering or lining of all internal and external body surfaces.

The terms “patient,” “subject” or “individual” interchangeably refers toa mammal, for example, a human or a non-human mammal, including primates(e.g., macaque, pan troglodyte, pongo), a domesticated mammal (e.g.,felines, canines), an agricultural mammal (e.g., bovine, ovine, porcine,equine) and a laboratory mammal or rodent (e.g., rattus, murine,lagomorpha, hamster).

The term “cellular and/or soluble macromolecular component” as usedherein refers to soluble substances constituting portions of the cell orproduced by cells, including cell membranes, cytosol, and solublemacromolecules (e.g. proteins, nucleic acids, polypeptides,glycoproteins, carbohydrates, lipids, phospholipids, etc.). Cellularand/or soluble macromolecular components that induce an immune responsein a subject are immunogenic antigens.

The term “recellularization” as used herein refers to removing the cellsfrom a biological tissue, for example, an organ, leaving only theextracellular matrix to be subsequently repopulated with cells,preferably live cells. Recellularization is especially useful in tissueengineering.

The term “extracellular matrix” (ECM) as used herein refers to theextensive and complex structure between the cells—the extracellular partof the biological tissue. The ECM generally comprises the structuralcomponent of the tissue, including its organization, shape, and strength(i.e., ability to resist external forces). Due to its diverse nature andcomposition, the ECM can serve many additional functions, such asproviding support and anchorage for cells, segregating tissues from oneanother, and regulating intercellular communication. The ECM caninfluence a cell's dynamic behavior. In addition, it sequesters a widerange of cellular growth factors and acts as a local depot for them.Included in the ECM are insoluble structural molecules that have beensecreted by cells and comprise components such as collagen, elastin, andlarge soluble proteoglycans.

A “decellularized extracellular matrix” refers to an ECM wherein theendogenous cells have been substantially removed. In variousembodiments, the decellularized ECM is isolated or separated from atleast about 60%, 70%, 80%, 90%, 95%, 99%, or more, of endogenouscellular material. The presence or extent of endogenous cellularmaterial can be determined using any method known in the art, e.g.,Western blotting, detection of nuclei, microscopy, etc.

The phrase “substantially intact” with respect to a tissue scaffoldand/or decellularized extracellular matrix (ECM) refers to an ECM thathas been subject to antigen removal and has structural integrity,biochemical composition and tensile strength that is not significantlydifferent from an ECM from the same tissue before it is subject toantigen removal.

The phrase “substantially free of endogenous antigens” with respect to atissue scaffold and/or decellularized extracellular matrix (ECM) refersto a tissue scaffold and/or ECM wherein the endogenous antigencomponents (e.g., proteins, lipids, carbohydrates, nucleic acids) havebeen substantially removed. In various embodiments, the decellularizedECM is isolated or separated from at least about 60%, 70%, 80%, 90%,95%, 99%, or more, of endogenous antigen components. The presence orextent of endogenous antigen components can be determined using anymethod known in the art, e.g., immunoassays, Western blotting, ELISA,gel electrophoresis, lymphocyte proliferation assays, etc. In variousembodiments, tissue scaffolds and/or ECM that are substantially free ofendogenous antigens do not elicit a significant or destructive immuneresponse, e.g., an allogeneic and/or xenogeneic immune response, invitro or in vivo, directed against the tissue scaffolds and/or ECM.

The term “sulfobetaine” refers to zwitterionic amphiphilic moleculesthat contain a polarized sulfobetaine head group (e.g.,dimethylsulfonioacetate (CH₃)₂S⁺—CH₂—CO₂ ⁻). In various embodiments, thehead group is followed by a three-carbon linkage between the quaternaryammonium and the amido nitrogen. In various embodiments, thesulfobetaine comprises a linear hydrocarbon tail composed of 13 to 16carbons. The sulfobetaine can be a detergent or a non-detergentmolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic of antigen removal (AR) and assessment ofresidual antigenicity. Residual hydrophiles (e.g., WSPs) and lipophiles(e.g., LSPs) extracted from bovine pericardium following AR (BP-AR) weresubjected to Western blot and probed with rabbit serum generated againstnative bovine pericardium. Residual hydrophilic and lipophilicantigenicity ratios were defined as the intensity of banding following 2days of AR divided by 1 min of AR.

FIG. 2 illustrates residual lipophilic antigenicity of bovinepericardium following one-step AR. Residual lipophilic antigenicity isnot significantly decreased with hydrophile solubilization (optimizedsolubilizing AR buffer (opt SARB) vs. basic AR buffer (BARB)) containingno additional additive, 134 mM 3-(benzyldimethylammonio)propanesulfonate (NDSB-256) or 0.1% (w/v) SDS. Results are plotted asmean±standard deviation. Groups not connected by the same letter aresignificantly different, p<0.05 (n=6 per group).

FIGS. 3A-C illustrate residual hydrophilic and lipophilic antigenicityand gross morphology of bovine pericardium following two-step AR.Hydrophilic antigenicity is not decreased further following addition ofa lipophile solubilization step (A). Lipophilic antigenicity issignificantly decreased following addition of lipophile solubilization(B). Results are plotted as mean±standard deviation. Groups notconnected by the same letter are significantly different, p<0.05 (n=6per group). Use of the Cordwell solution in opt SARB or 1% (w/v) SDS inBARB dramatically alters gross tissue morphology (C). The scale barrepresents 1 cm.

FIGS. 4A-B illustrate residual lipophilic antigenicity and grossmorphology of bovine pericardium following two-step AR with either theentire Cordwell solution or its individual components. Use of opt SARBcontaining 8 M urea and 2 M thiourea, 1% (w/v) ASB-14 or the entireCordwell solution significantly reduces residual lipophilic antigenicitycompared to opt SARB alone (A). Results are plotted as mean±standarddeviation. Groups not connected by the same letter are significantlydifferent, p<0.05 (n=6 per group). Treatment with 8 M urea and 2 Mthiourea in opt SARB or the entire Cordwell solution drastically changesgross tissue morphology (B). The scale bar represents 1 cm.

FIGS. 5A-B illustrate tensile properties of bovine pericardium (BP).Young's modulus (A) and UTS (B) of BP following two-step AR using noadditive or 1% (w/v) ASB-14 in opt SARB, or 1% (w/v) SDS in basic ARbuffer are not significantly different from those of native BP. A secondstep of AR using 8 M urea and 2 M thiourea in opt SARB results in asignificant decrease in Young's modulus and UTS. Results are plotted asmean±standard deviation. Groups not connected by the same letter aresignificantly different, p<0.05 (n=6 per group).

FIGS. 6A-D illustrate quantitative biochemical analysis of bovinepericardium (BP) composition. Water content is maintained followingtwo-step AR in opt SARB containing no additional additive or 1% (w/v)ASB-14 compared to native BP (A). A second step of AR using either 8 Murea and 2 M thiourea in opt SARB or 1% (w/v) SDS in BARB significantlyincreases water content. The collagen content per dry weight (DW) is notsignificantly different following two-step AR compared to native BP (B).The elastin content per DW is maintained following two-step AR using optSARB containing no additional additive or 1% (w/v) ASB-14 compared tonative BP (C). Use of 8 M urea and 2 M thiourea in opt SARBsignificantly decreases the elastin content per DW. The elastin contentper DW for samples treated with 1% (w/v) SDS in BARB is below the limitof detection of the assay. GAG content per DW is significantly decreasedfollowing two-step AR compared to native BP (D). Results are plotted asmean±standard deviation. Groups not connected by the same letter aresignificantly different, p<0.05 (n=6 per group).

FIG. 7 illustrates gross histological morphology and residual knownxenoantigens in representative images of bovine pericardium (BP). H&Estaining reveals both preservation of histological ECM morphology andreduction in residual nuclei following two-step AR using 1% (w/v) ASB-14in opt SARB. Treatment with 8 M urea and 2 M thiourea in opt SARB or 1%(w/v) SDS in BARB does not maintain histological ECM morphology.Verhoeff van Gieson staining indicates that gross collagen and elastinstructure is preserved following two-step AR using no additive or 1%(w/v) ASB-14 in opt SARB for lipophile solubilization. Treatment with 8M urea and 2 M thiourea in opt SARB or 1% (w/v) SDS in BARB does notmaintain gross collagen and elastin organization. Immunohistochemicalstaining reveals that no α-gal antigens persist in BP treated with 1%(w/v) ASB-14 in opt SARB or 1% (w/v) SDS in BARB. Residual α-galantigens are observed in BP subjected to no additive or 8 M urea and 2 Mthiourea in opt SARB. Immunohistochemical staining indicates that no MHCI antigens persist in BP treated with 1% (w/v) ASB-14 in opt SARB.Residual MHC I antigens are observed in BP subjected to no additive or 8M urea and 2 M thiourea in opt SARB or 1% (w/v) SDS in BARB. The scalebar represents 50 μm.

FIG. 8 illustrates residual nuclei per HPF in bovine pericardium (BP).Following two-step AR, 1% (w/v) ASB-14 in opt SARB or 1% (w/v) SDS inBARB reduces nuclei most significantly compared to native BP. Resultsare plotted as mean±standard deviation. Groups not connected by the sameletter are significantly different, p<0.05 (n=6 per group).

FIG. 9 illustrates fluorescence appearance of bovine pericardium (BP)recellularized with eGFP transfected human mesenchymal stem cells(hMSC). Native group is composed of untreated BP, optSARB (100 mM DTTplus 100 mM KCl) group is treated for removal of water-soluble antigensonly using the solubility methods described in this patent application,ASB-14 group comprises both removal of water-soluble antigens (usingoptSARB) plus lipid-soluble antigens using ASB-14 as described in thepresent methods, SDS group is treated with 1% SDS which represents thecurrent most commonly utilized literature control method fordecellularization of xenogeneic tissue. Note that recellularizationefficiency, as demonstrated by presence of eGFP-expressing hMSCs isunchanged between the native, optSARB and ASB-14 groups. In comparison,SDS group is incapable of supporting recellularization with eGFP labeledhMSCs (note complete absence of green fluorescence).

FIG. 10 illustrates histological analysis of bovine pericardium (BP)explants following one week of implantation in New Zealand Whiterabbits. Native group consists of native BP, optSARB group is treatedfor removal of water-soluble antigens only using the solubility methodsdescribed in this patent application, ASB-14 group comprises bothremoval of water-soluble antigens (using optSARB) plus lipid-solubleantigens using ASB-14 as described in the present methods, SDS group istreated with 1% SDS which represents the current most commonly utilizedliterature control method for decellularization of xenogeneic tissue,fixed group consists of commercially available (St Jude medical)glutaraldehyde-fixed BP which is a currently licensed FDP approved heartvalve and vessel patch material. All groups demonstrate mild to moderateinflammatory cell infiltration. Scale bar represents 500 μm.

FIG. 11 illustrates histological analysis of bovine pericardium (BP)explants following six weeks of implantation in New Zealand Whiterabbits. Native group consists of native BP, optSARB group is treatedfor removal of water-soluble antigens only using the solubility methodsdescribed in this patent application, ASB-14 group comprises bothremoval of water-soluble antigens (using optSARB) plus lipid-solubleantigens using ASB-14 as described in the present methods, SDS group istreated with 1% SDS which represents the current most commonly utilizedliterature control method for decellularization of xenogeneic tissue,fixed group consists of commercially available (St Jude medical)glutaraldehyde-fixed BP which is a currently licensed FDP approved heartvalve and vessel patch material. A minimal amount of small mononuclearcells (MNCs) are observed in the subdermal, subpannicular, andperi-scaffold regions associated with ASB-14 treated BP-AR. A mild levelof small MNCs is associated with SDS-decellularized BP. A moderateamount of small MNCs were found with fixed BP or native BP. A severelevel of small MNCs, including formation of lymphoid follicles, isassociated with opt SARB alone-treated BP-AR. Stepwise antigen removalusing ASB-14 decreases the amount of MNCs in the peri-scaffold regioncompared to native BP, opt SARB-treated BP, SDS-decellularized BP, orfixed BP. Little to no fibrous encapsulation is seen with ASB-14 treatedBP-AR, opt SARB-treated BP-AR, or native BP. Conversely, notable fibrousencapsulation is associated with SDS-decellularized BP or fixed BP.Stepwise antigen removal using ASB-14 is associated with less fibrousencapsulation than SDS-decellularized BP or fixed BP. Finally, markedcellular ingrowth is observed with ASB-14 treated BP-AR or optSARB-treated BP-AR. However, minimal cellular ingrowth is associatedwith SDS-decellularized BP or fixed BP. Stepwise antigen removal usingASB-14 supports robust cellular repopulation of BP-AR. In contrast toclinically-approved fixed BP or SDS-decellularized BP: (1) Less smallMNCs are elicited to respond to ASB-14 treated BP-AR at six weeksfollowing implantation. (2) ASB-14 treated BP-AR has not been walled offby fibrous encapsulation in the rabbit. (3) ASB-14 treated BP-AR is ableto support recellularization with host cells. Taken together, thesefindings suggest indicate that stepwise, solubilization-based antigenremoval using optSARB followed by ASB-14 reduces the in vivo immuneresponse towards BP-AR compared to native BP; BP following one-step,solubilization-based antigen removal (opt SARB); SDS-decellularized BP;and clinically approved gluraraldehyde-fixed BP. Scale bar represents500 μm.

FIG. 12 illustrates Histologic sections of heart muscle scaffoldsgenerated using the protocols described herein. Top Row: NativeLV—native unprocessed rat left ventricular (LV) heart muscle tissue.Second Row: Following hydrophilic antigen removal alone usingsolubilization antigen removal buffer (SARB, containing DTT and KCl) for2 days, cardiomyocytes show evidence of mild cytoplasmic and nuclearvacuolation. Third Row: Addition of Latrunculin to SARB protocol resultsin increased coalescing areas of cytoplasmic vacuolation, milddisruption of sarcomeric structure and almost complete removal ofnuclei. Fourth and Fifth Rows: Addition of ASB-14 (hydrophobicsolubilization) prior to application of Latrunculin/SARB results incomplete removal of some cardiomyocytes while retaining normal structureof the extracellular matrix (ECM) in these regions, although somecardiomyocytes remain. Sixth Row: Relaxation of the myocardium prior toapplication of the 1% ASB-14, Latrunculin, SARB sarcomere disassemblyand solubilization protocol, results in a significant increase in theproportion of cardiomyocytes which are completely removed by theprotocol, again ECM structure is retained, although some cardiomyocytesremain. Seventh Row: Relaxation of the myocardium followed byapplication of 2% ASB-14, Latrunculin and SARB results in completeremoval of all cardiomyocytes and nuclei from the material. ECM matrixstructure is completely retained.

FIG. 13 illustrates reduced antigenicity of left ventricular myocardialtissue. Biomaterial residual antigenicity was assessed using Westernblot methods published in the scientific literature and describedherein. a; Western blot of residual antigens extracted from myocardialpatches (MP) probed using mouse anti-rat LV poly-polyclonal serum.Lanes: 1; MagicMark Protein Ladder (Invitrogen), 2; BARB native tissuecontrol, 3; optSARB, 4; 1% SDS, 5; myocardial patch following antigenremoval (MP-AR) as described in the current invention (Step 1 forWater-Soluble antigens: LatrunculinB, KCl, KI; followed by Step 2 forLipid-Soluble antigens: 2% ASB in optSARB), 6; MP-AR using 3% ASB inoptSARB in the second step of AR, 7; MP-AR using 4% ASB in optSARB, b;Densitometry analysis for residual antigenicity. Results displayed asmeans±standard deviation. Our antigen removal approach (MP-AR) resultsin removal of >90% of antigens from the myocardial patch. Groups notconnected by the same letter are significantly different, p<0.01 (n=6per group).

DETAILED DESCRIPTION 1. Introduction

The present invention is based, in part, on methods for the sequentialsolubilization and extraction of water-soluble antigenic components andlipid-soluble antigenic components from tissue to produce tissuescaffold or decellularized extracellular matrix (ECM) with ECMstructure, biochemical composition, mechanical properties, andrecellularization capacity that is substantially the same as the tissueprior to antigen removal procedures. Stepwise or sequential antigenremoval of water-soluble and lipid-soluble antigens from tissuesubstantially reduce in vivo immune response towards the produced tissuescaffold or decellularized ECM. Sequential solubilization and extractionof water-soluble antigenic components and lipid-soluble antigeniccomponents from tissue allows for the production of animmunologically-acceptable, structurally-integral, mechanically-sound,tissue scaffold compatible with recellularization, e.g., for use intissue engineering and/or tissue regeneration.

The present invention is further based, in part, on methods for thesequential solubilization and extraction of cytoskeletal components(e.g., filamentous actin and/or microtubules), water-soluble antigeniccomponents and lipid-soluble antigenic components from tissue to producetissue scaffold or decellularized extracellular matrix (ECM) with ECMstructure, biochemical composition, mechanical properties andrecellularization capacity that is substantially the same as the tissueprior to antigen removal procedures. Stepwise or sequential antigenremoval of cytoskeletal components, water-soluble and lipid-solubleantigens from tissue substantially reduces in vivo immune responsetowards the produced tissue scaffold or decellularized ECM. Sequentialsolubilization and extraction of cytoskeletal components, water-solubleantigenic components and lipid-soluble antigenic components from tissueallows for the production of an immunologically-acceptable, structurallyintegral, mechanically sound, tissue scaffold compatible withrecellularization, e.g., for use in tissue engineering and/or tissueregeneration. When applied to muscle tissues, the methods can furthercomprise the step of relaxing, solubilizing and removing sarcomericcomponents.

Biomaterial antigenicity is the primary hurdle for the use of xenogeneicscaffolds in tissue engineering and regenerative medicine. To reduce oreliminate the persistent presence of antigens on decellularized tissueto elicit the immune response upon implantation we have developed a morerigorous antigen removal (AR) process. Solubilization-based AR has beenshown to enhance the removal of water-soluble protein (WSP) antigens intissues, including bovine pericardium (BP), beyond that achieved bydecellularization using hypotonic solution or SDS (e.g., at aconcentration in the range of 0.1-1.0% w/v). However, the diversity ofprotein antigens within a tissue necessitates development of ARstrategies capable of addressing a spectrum of protein antigensolubilities. The present invention is based, in part, on the discoveryof antigen removal (AR) methods promoting the solubilization oflipid-soluble proteins (LSPs) to reduce the residual LSP antigenicity oftissues, including BP, when applied as a separate step of AR tosolubilizing WSP. Promoting the solubilization of a protein subset(cytoskeletal components, WSPs and/or LSPs) significantly reduces theresidual antigenicity of that specific subset of protein antigens(cytoskeletal components, WSPs and/or LSPs, respectively). However,promoting the solubilization of a protein subset (cytoskeletalcomponents, WSPs and/or LSPs) does not significantly reduce residualantigenicity of the other subset of protein antigens (cytoskeletalcomponents, WSPs and/or LSPs, respectively). Facilitating solubilizationof cytoskeletal components (e.g., by including one or more cytoskeletaldestabilizing and/or depolymerizing agents), WSP (e.g., using 100 mMdithiothreitol and 100 mM potassium chloride in solubilizing antigenremoval buffer) and LSP (e.g., using 1-4% (w/v) ASB-14 in solubilizingantigen removal buffer) in a multi-step sequential, differential ARstrategy markedly reduces the residual antigenicity of tissues beyondthat achieved with either one-step AR or decellularization by 1% (w/v)SDS. Use of 1-4% (w/v) ASB-14 for LSP AR does not compromise thebiomaterial properties of tissue following antigen removal. A multi-stepAR strategy promoting sequential, differential protein solubilizationsignificantly reduced residual LSP antigens beyond that achieved withone-step AR of WSPs while maintaining biomaterial functional properties.Moreover, total DNA content and/or residual nuclei counts may not be anappropriate indicator of residual LSP antigenicity. This studydemonstrates the importance of a sequential, differential proteinsolubilization approach for the reduction of biomaterial antigenicity inxenogeneic scaffold generation for tissue engineering.

For the application to muscle tissue, principles of protein chemistrycan be applied to antigen removal of each antigenic class from muscletissue. The methods entail a multimodal approach to disassemble andsolubilize the myocyte sarcomere structure (the functional rigidforce-producing units that make up myocytes). This approach producesmore complete removal of antigens from myocytes than previously reportedmethods, while maintaining ECM structure-function relationships andimproving recellularization capacity. The methods described hereinproduce decellularized tissue and ECM scaffolds with significantlyreduced antigenicity, due to more complete removal of cytoskeletal,sarcomeric, hydrophilic and lipophilic antigens. This present methodsemploy macromolecular disassembly of the basic structural unit ofmyocytes (sarcomeres) combined with the principles of sequential,differential solubilization of water-soluble antigens and lipid-solubleantigens for sequential myocyte solubilization and antigen removal frommuscle tissue (e.g., cardiac muscle tissue, striated or skeletal muscletissue, smooth muscle tissue).

With respect to muscle tissue, the methods generally involve thetreatment of allogeneic (e.g., from a different individual of the samespecies of the subject receiving the tissue) and xenogeneic (e.g., froma different species of the subject receiving the tissue) muscle tissues(e.g., myocardial, skeletal or smooth muscle) for the purpose ofremoving cells, cellular debris, antigens, proteins, nucleic acids,phospholipids, and other macromolecules prior to implantation. Theapproach to production of such ECM scaffolds described herein is basedon the protein chemistry principles of sequential, differentialsolubilization, utilized for protein extraction from homogenizedtissues, e.g., for proteomic applications. Protein solubilization isknown to be important for achieving protein extraction from homogenizedsample material for use in subsequent two-dimensional gelelectrophoresis (2-DE). Protein precipitation tends to occur due tomolecular interactions which result in protein aggregation (e.g.,disulfide bond formation, hydrophobic interactions, non-covalentinteractions). Consequently, disruption of macromolecular interactionswithin and between proteins is important for achieving and maintainingprotein solubility. Therefore, the goal of most protein extractionprotocols is to disaggregate, denature, reduce and consequentiallysolubilize the protein type of interest. Furthermore, various proteintypes within a tissue exhibit different physicochemical properties(e.g., cytoplasmic proteins are generally water soluble, integralmembrane proteins are generally hydrophobic). No single extractioncondition is capable of simultaneously solubilizing all proteins withina tissue.

Protein extraction protocols, therefore, generally utilize sequentialextraction techniques to solubilize proteins in a stepwise manner fromthe tissue by serial application of extraction solutions withcharacteristics designed to favor solubilization of a particular proteintype. Previously reported decellularization and AR methods have largelyfailed to apply the principles of protein chemistry required to achievesolubilization of various antigenic protein types within the tissue. Thepresent methods are based, in part, on the recognition that themacromolecular structure of the sarcomere prevents solubilization andsubsequent removal of these components from the material. We thereforedesigned a specifically targeted stepwise approach to relax,depolymerize and render the components of the sarcomere amenable tosolubilization. Combining sarcomere disassembly and solubilization withsequential, differential solubilization of water soluble antigens andlipid soluble antigens for antigen removal results in enhanced removalof cellular components and antigens from muscle tissues.

2. Methods for Removing Antigen Components from Tissue

The present methods relate to the removal of antigens from tissues,e.g., cells, cellular debris, proteins, nucleic acids, phospholipids,carbohydrates, and other macromolecules, e.g., from tissues allogeneic(i.e., derived from within the same species) to or xenogeneic (i.e.,derived from different species) to a recipient of the tissue, e.g., intissue or organ transplantation or regeneration.

a. Tissues

The tissues subject to sequential antigen removal can be from any tissuesuitable for transplantation. Generally, the tissue is live and unfixed.In various embodiments, tissues subject to sequential antigen removalinclude without limitation connective tissue, muscle tissue, nervoustissue (of the brain, spinal cord, and nerves), epithelial tissue, andorgan tissue. Connective tissue includes fibrous tissue like fascia,tendon, ligaments, heart valves, bone, and cartilage. Muscle tissueincludes skeletal muscle tissue, smooth muscle tissue, such asesophageal, stomach, intestinal, bronchial, uterine, urethral, bladder,and blood vessel tissue, and cardiac muscle tissue. Epithelial tissueincludes simple epithelial tissue, such as alveolar epithelial tissue,blood vessel endothelial tissue, and heart mesothelial tissue, andstratified epithelial tissue. In various embodiments, the tissue issubject to antigen removal is heart, heart valve, vessel, vascularconduit, artery, vein, skin, dermis, pericardium, dura, intestinalsubmucosa, ligament, tendon, bone, cartilage, ureter, urinary bladder,liver, lung, and umbilical cord. The tissue may be a part of an organ orbe an intact organ.

The tissue may be from the intended recipient (e.g., is syngeneic), fromthe same species as the intended recipient (e.g., is allogeneic) or froma different species from the intended recipient (e.g., is xenogeneic) ofthe tissue scaffold or decellularized ECM produced by removal of theantigens. In various embodiments, the tissue is from a first human andintended to be transplanted into second human. In various embodiments,the tissue is from a porcine, ovine, bovine, ostrich (e.g., of the genusStruthio) or a non-human primate and intended to be transplanted into ahuman.

As appropriate, the tissue can be submerged in solubilization solutionor perfused with solubilizing solution. Antigens can be effectivelyremoved from thinner tissues (e.g., tissues having less about 1 mmthickness) by submerging. Antigens can be effectively removed fromthicker tissues and intact organs by perfusion of the tissue withsolubilizing solution.

Generally, the tissue is subjected to solubilization as soon aspracticable after extraction from the original host and before thetissue is substantially decomposed. In various embodiments, the tissueis subjected to solubilization within 12 hours after extraction, e.g.,within 10, 8, 6, 4, 3, 2, 1 hours after extraction from the originalhost.

b. Destabilizing and/or Depolymerizing Cytoskeletal Components

In various embodiments, the tissue can be contacted with (e.g.,submerged in or perfused with) a solution for destabilizing and/ordepolymerizing one or more cytoskeletal components (e.g., filamentousactin and/or microtubules) under sufficient conditions and for asufficient time to depolymerize, solubilize and extract a portion of thecytoskeletal components, e.g., polymerized and/or filamentouscytoskeletal components. In various embodiments, the tissue is contactedwith one or more agents that destabilize and/or depolymerize filamentousactin and/or microtubules.

In some embodiments, the one or more cytoskeletal destabilizing and/ordepolymerizing agents comprise one or more actin destabilizing and/ordepolymerization agents. In some embodiments, the one or more actindestabilizing and/or depolymerizing agents are selected from the groupconsisting of Cytochalasin A, Cytochalasin B, Cytochalasin C,Cytochalasin D, Cytochalasin E, Cytochalasin F, Cytochalasin G,Cytochalasin H, Cytochalasin I, Cytochalasin J, Latrunculin A,Latrunculin B, Swinholide A, Misakinolide A, Bistheonelide A,Scytophycin A, Scytophycin B, Scytophycin D, Scytophycin E,19-O-Demethylscytophycin C, 6-Hydroxyscytophycin B,6-Hydroxy-7-O-methylscytophycin E and tolytoxin, Mycalolide A,Mycalolide B, Mycalolide C, secomycalolide A and 30-hydroxymycalolide A,Halichondramide, (19Z)-halichondramide, kabiramides B, kabiramides C,kabiramides D, kabiramides G, kabiramides J, kabiramides K, ulapualideA, jaspamide, Dihydrohalichondramide, Aplyronine A, Aplyronine B,Aplyronine C, Pectenotoxin 2, Pectenotoxin 6, and Migrastatin. In someembodiments, the actin depolymerization agents depolymerize filamentouscytoskeletal actin (F-actin). In some embodiments, the actindepolymerization agents depolymerize filamentous α-sarcomeric actin(F-actin).

In some embodiments, the one or more cytoskeletal destabilizing and/ordepolymerizing agents comprise one or more microtubule depolymerizationand/or destabilizing agents. In some embodiments, the one or moremicrotubule depolymerization and/or destabilizing agents is selectedfrom the group consisting of colchicine, colcemid, vinblastine,vincristine, myoseverin, nocodazole, podophyllotoxin, polygamain andtaxol.

In various embodiments, the solution comprising the one or morecytoskeletal component destabilizing and/or depolymerizing agents is anaqueous solution. In varying embodiments, the solution comprising theone or more cytoskeletal component destabilizing and/or depolymerizingagents is a physiologically isotonic aqueous solution (e.g., serum freecell culture solution). In varying embodiments, the one or morecytoskeletal component destabilizing and/or depolymerizing agents isadded to a solution for solubilizing the water-soluble antigens, asdescribed below, in the section entitled “Solubilizing Water-SolubleAntigens in the Tissue.” In some embodiments, the aqueous solutioncomprises glucose, e.g., at a concentration in the range of about 5-50mM, e.g., about 5-25 mM, e.g., about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM,30 mM, 35 mM, 40 mM, 45 mM, or 50 mM.

In varying embodiments, the tissue is contacted with (e.g., is submergedin or perfused with) each cytoskeletal destabilizing and/ordepolymerizing agent at a concentration in the range of about 1 nM toabout 10 μM, e.g., at a concentration of about 1 nM, 5 nM, 10 nM, 25 nM,50 nM, 75 nM, 100 nM, 500 nM, 750 nM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM,25 μM, 30 μM, 35 μM, 40 μM, 45 μM and 50 μM.

In various embodiments, the tissue is submerged in or perfused with thesolution comprising one or more cytoskeletal depolymerizing and/ordestabilizing agents for at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22 or 24 hours, e.g., for at least about 2, 3, 4, 5, 6, 7days, as appropriate. The tissue may be washed one or more times duringthe time period of submerging or perfusing, e.g., to promote diffusionand separation of cytoskeletal antigens from the tissue.

In various embodiments, the depolymerization, solubilization, extractionand/or removal of depolymerized cytoskeletal components is performed ata temperature above freezing (e.g., above 0° C.) and at or below bodytemperature (e.g., at or below about 37° C.). In various embodiments,the depolymerization, solubilization, extraction and/or removal ofdepolymerized cytoskeletal components is performed at a refrigeratedtemperature, e.g., between about 4-10° C. In various embodiments, thedepolymerization, solubilization, extraction and/or removal ofdepolymerized cytoskeletal components is performed at room temperature,e.g., between about 20-30° C., e.g., about 25° C. In variousembodiments, the depolymerization, solubilization, extraction and/orremoval of depolymerized cytoskeletal components is performed at humanbody temperature, e.g., about 37° C. The depolymerization,solubilization, extraction and/or removal steps can be performed at thesame or different temperatures.

In performing the methods, the step of contacting the tissue with asolution comprising one or more cytoskeletal destabilizing and/ordepolymerizing agents can be conducted concurrently with or in aseparate step from the step of contacting the tissues with a solutionfor solubilizing the water-soluble antigens. After a portion of thecytoskeletal components are depolymerized, solubilized and extractedfrom the tissue, the solubilized and extracted cytoskeletal componentsare separated from the tissue, e.g., in a wash step. In variousembodiments, washing is performed by contacting tissue (e.g., submergingin or perfusing with) with fresh physiologically isotonic solution(e.g., serum free cell culture solution) or a solution for solubilizingthe water-soluble antigens, as described below. The step ofdepolymerizing, solubilizing and extracting cytoskeletal components canbe performed for one or multiple iterations, e.g., 2, 3, 4, 5, or moreiterations, as appropriate. For example, the iterations of solubilizingdepolymerized cytoskeletal components and separating (e.g., rinsing) thecytoskeletal components from the tissue can be repeated until extractedcytoskeletal components are no longer detected in the depolymerizingsolution or the tissue, or until the detectable cytoskeletal componentsin the extraction solution or the tissue fall below a predeterminedthreshold level, as appropriate.

c. Solubilizing Water-Soluble Antigens in the Tissue

The tissue can be contacted with (e.g., submerged in or perfused with) asolution for solubilizing the water-soluble antigens under sufficientconditions and for a sufficient time to extract a portion of the watersoluble antigens from the tissue or to reach an equilibrium between thewater-soluble antigens within the tissue and water soluble antigens inthe solution, as appropriate.

In various embodiments, the tissue is submerged in or perfused with thesolution for solubilizing the water-soluble antigens for at least about0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours, e.g., for atleast about 2, 3, 4, 5, 6, 7 days, as appropriate. The tissue may bewashed one or more times during the time period of submerging orperfusing, e.g., to promote diffusion and separation of water-solubleantigens from the tissue.

In various embodiments, removal of water-soluble antigens is performedat a temperature above freezing (e.g., above 0° C.) and at or below bodytemperature (e.g., at or below about 37° C.). In various embodiments,removal of water-soluble antigens is performed at a refrigeratedtemperature, e.g., between about 4-10° C. In various embodiments,removal of water-soluble antigens is performed at room temperature,e.g., between about 20-30° C., e.g., about 25° C. In variousembodiments, removal of water-soluble antigens is performed at humanbody temperature, e.g., about 37° C.

In various embodiments, the water-soluble antigens are solubilized in asolution comprising a buffering agent, a reducing agent, a proteaseinhibitor, and one or more salts suitable for maintaining proteinsolubility.

In various embodiments, the buffering agent maintains a pH (e.g., has apKa) to allow for solubility of the antigens in aqueous solution. Forexample, in some embodiments, the buffering agent maintains a pH in therange of about 4-11, e.g., a pH in the range of about 4-6, 8-11, 5-10 or6-9. In some embodiments, the buffering agent maintains a pH of at leastabout 8. In some embodiments, the buffering agent maintains a pH of lessthan about 5. Illustrative buffering agents include without limitationTris-HCl, phosphate, citric acid, acetate, imidazole, carbonate, MES,Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES,HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine,Gly-Gly, Bicine, HepBS, TAPS, AMPD, CHES, CAPSO, AMP, CAPS and CABS.These and other buffering agents of use are well-known in the art andcommercially available, e.g., from Sigma-Aldrich (on the internet atsigmaaldrich.com). In some embodiments, the buffering is Tris-HCl.

In some embodiments, the one or more salts comprise a monovalent or adivalent anion. In some embodiments, the one or more salts comprise ametal halide salt. Illustrative metal halide salts of use includewithout limitation LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl,KBr, KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂,BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂,SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂, BaI₂, and mixtures thereof. In someembodiments, the one or more salts comprise KCl. In various embodiments,the one or more salts are included at a concentration of at least about50 mM, 75 mM, or 100 mM, for example, at least about 150 mM, 200 mM, 250mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM. 600 mM, 700 mM, 800 mM, 900mM or 1000 mM, for example, in the range of about 100-500 mM or about100-200 mM.

Illustrative reducing agents for use in the solution for solubilizingthe water-soluble antigens include without limitation Tributylphosphine(TBP), beta mercaptoethanol, 2-mercaptoethylamine,tris(2-carboxyethyl)phosphine (TCEP), cysteine-HCl, and dithiothreitol(DTT). In some embodiments, the reducing agent is DTT.

Illustrative protease inhibitors for use in the solution forsolubilizing the water-soluble antigens include without limitationaspartic protease inhibitors, cysteine protease inhibitors,metalloprotease inhibitors, serine protease inhibitors (serpins),threonine protease inhibitors, trypsin inhibitors, and mixtures thereof.In various embodiments, the protease inhibitor is an 19, 110, 114, 124,129, 134, 136, 142, 148, 153, 167, 168, 178 inhibitor, or a mixturethereof. In some embodiments, the protease inhibitor is AEBSF(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), also sold asPEFABLOC®. In some embodiments, the protease inhibitor isPhenylmethylsulfonyl fluoride (PMSF). Numerous protease inhibitorcocktails of use are commercially available from Roche MolecularBiochemicals.

In some embodiments, the water-soluble antigens are solubilized in asolution that comprises one or more of an antibacterial agent and/or anantifungal agent.

In some embodiments, the water-soluble antigens are solubilized in asolution that comprises a chelation agent. Illustrative chelation agentsinclude without limitation ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), Citric Acid,N,N-bis(carboxymethyl)glycine (NTA), and the meso isomer ofDimercaptosuccinic acid (DMSA).

In some embodiments, the water-soluble antigens are solubilized in asolution that does not comprise an amphiphile. In some embodiments, thewater-soluble antigens are solubilized in a solution that does notcomprise a detergent. In some embodiments, the water-soluble antigensare solubilized in a solution that comprises a non-detergentsulfobetaine. Illustrative non-detergent sulfobetaines include withoutlimitation NDSB-256, NDSB-211, NDSB-195, NDSB-221 and NDSB-201.

In some embodiments, the water-soluble antigens are solubilized in asolution comprising Tris-HCl, dithiothreitol (DTT), a proteaseinhibitor, and KCl. In a particular embodiment, the water-solubleantigens are solubilized in a solution comprising 10 mM Tris-HCl, 100 mMDTT, 100 mM KCl and 2 mM MgCl₂. A protease inhibitor and/or anantibacterial agent and/or an antifungal agent may also be included.

d. Separating the Tissue from the Solubilized Water-Soluble Antigens

Once the tissue has been submerged in or perfused with the solution forsolubilizing the water-soluble antigens under sufficient conditions andfor a sufficient time, the tissue can be separated from the solution,now containing a portion of the water soluble antigens extracted fromthe tissue. Separation of the tissue and the solubilizing solution canbe performed using any methods known in the art. In various embodiments,the tissue can be rinsed, e.g., in the same solution for solubilizingthe water-soluble antigens. Optionally, the tissue can be subject tosaturation with (e.g., submerged in or perfused with) solubilizationsolution and separated for one or more iterations in fresh solution forsolubilizing the water-soluble antigens for a sufficient time to extracta further portion of the water soluble antigens from the tissue or toreach an equilibrium between the water-soluble antigens within thetissue and water soluble antigens in the solution, as appropriate. Theiterations of submerging in or perfusing with solution for solubilizingthe water-soluble antigens and separating (e.g., rinsing) can berepeated until extracted water-soluble antigens are no longer detectedin the solution for solubilizing the water-soluble antigens, or untilthe detectable extracted water-soluble antigens fall below apredetermined threshold level, as appropriate.

In various embodiments, the separation and removal of the water-solubleantigens from the tissue can involve centrifugation and/or filtration,as appropriate, to separate the tissue from the solution forsolubilizing the water-soluble antigens containing a portion of and/orsaturated with extracted water-soluble antigens. In other embodiments,the tissue is removed from solution for solubilizing the water-solubleantigens and rinsed with being subjected to centrifugation orfiltration.

In various embodiments, more frequent iterations of submerging orperfusing and separation (e.g., rinsing) and a larger number ofiterations of submerging or perfusing and separating (e.g., rinsing) canallow for faster solubilization and removal of water-soluble antigensfrom the tissue. For example, the tissue can be saturated with (e.g.,submerged in or perfused with) a first solution for solubilizing thewater-soluble antigens for a sufficient time to extract at least aportion of the water soluble antigens; separated from the firstsolution, now containing a portion of extracted water soluble antigens,before equilibrium is reached between the water-soluble antigens withinthe tissue and water soluble antigens in the solution; saturated with(e.g., submerged in or perfused with) a second solution for solubilizingthe water-soluble antigens for a sufficient time to extract at least aportion of the water soluble antigen; and separated from the secondsolution, now containing a portion of extracted water soluble antigens,before equilibrium is reached between the water-soluble antigens withinthe tissue and water soluble antigens in the solution. Furtheriterations of submerging or perfusing and separation can be performed,as appropriate or desired, until a sufficiently low level of watersoluble antigens extracted into the solution for solubilizing thewater-soluble antigens is achieved.

e. Solubilizing Lipid-Soluble Antigens in the Tissue

In various embodiments, tissues that have been subject to extraction andseparation of water-soluble antigens can subsequently be subject toextraction of lipid-soluble antigens. In other embodiments, tissues arefirst subject to extraction of lipid soluble antigens and second subjectto extraction of water-soluble antigens.

The tissue can be saturated with (e.g., submerged in or perfused with) asolution for solubilizing the lipid-soluble antigens for a sufficienttime to extract a portion of the lipid soluble antigens from the tissueor to reach an equilibrium between the lipid-soluble antigens within thetissue and lipid soluble antigens in the solution, as appropriate.

In various embodiments, the tissue is saturated with (e.g., submerged inor perfused with) the solution for solubilizing the lipid-solubleantigens for at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22 or 24 hours, e.g., for at least about 2, 3, 4, 5, 6, 7 days, asappropriate. The tissue may be washed one or more times during the timeperiod of submerging or perfusing, e.g., to promote diffusion andseparation of lipid-soluble antigens from the tissue.

In various embodiments, removal of lipid-soluble antigens is performedat a temperature above freezing (e.g., above 0° C.) and at or below bodytemperature (e.g., at or below about 37° C.). In various embodiments,removal of lipid-soluble antigens is performed at a refrigeratedtemperature, e.g., between about 4-10° C. In various embodiments,removal of lipid-soluble antigens is performed at room temperature,e.g., between about 20-30° C., e.g., about 25° C. In variousembodiments, removal of lipid-soluble antigens is performed at humanbody temperature, e.g., about 37° C.

In some embodiments, the lipid-soluble antigens are solubilized in asolution comprising a buffering agent, a reducing agent, a proteaseinhibitor, one or more salts suitable for maintaining protein solubilityand an amphiphile.

In various embodiments, the amphiphile is a zwitterionic detergent. Insome embodiments, the amphiphile is a sulfobetaine. Illustrativesulfobetaines of use include without limitation3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(amidosulfobetaine-14; ASB-14); amidosulfobetaine-16 (ASB-16);4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine (ASB-C8Ø);3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB3-8);N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10),N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-12),N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-14);3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16);3-(N,N-Dimethyloctadecylammonio) propanesulfonate (SB3-18);3-(1-Pyridinio)-1-propanesulfonate (NDSB-201); 3-(Benzyldimethylammonio)propanesulfonate (NDSB-256); NDSB-211, NDSB-195, NDSB-221;3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), and mixtures thereof. In various embodiments, the sulfobetaineis an amidosulfobetaine. Illustrative amidosulfobetaines include withoutlimitation, ASB-14, ASB-16 and ASB-C8Ø. In various embodiments, thesulfobetaine is a non-detergent sulfobetaine. Illustrative non-detergentsulfobetaines include without limitation NDSB-256, NDSB-211, NDSB-195,NDSB-221 and NDSB-201.

In various embodiments, the buffering agent maintains a pH (e.g., has apKa) to allow for solubility of the antigens in aqueous solution. Forexample, in some embodiments, the buffering agent maintains a pH in therange of about 4-11, e.g., a pH in the range of about 4-6, 8-11, 5-10 or6-9. In some embodiments, the buffering agent maintains a pH of at leastabout 8. Illustrative buffering agents include without limitationTris-HCl, phosphate, citric acid, acetate, imidazole, carbonate, MES,Bis-Tris, ADA, aces, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES,HEPES, DIPSO, MOBS, TAPSO, Trizma, HEPPSO, POPSO, TEA, EPPS, Tricine,Gly-Gly, Bicine, HepBS, TAPS, AMPD, CHES, CAPSO, AMP, CAPS and CABS.These and other buffering agents of use are well-known in the art andcommercially available, e.g., from Sigma-Aldrich (on the internet atsigmaaldrich.com). In some embodiments, the buffering is Tris-HCl.

In some embodiments, the one or more salts comprise a monovalent or adivalent anion. In some embodiments, the one or more salts comprise ametal halide salt. Illustrative metal halide salts of use includewithout limitation LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl,KBr, KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂,BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂,SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂, BaI₂, and mixtures thereof. In someembodiments, the one or more salts comprise KCl. In various embodiments,the one or more salts are included at a concentration of at least about50 mM, 75 mM, or 100 mM, for example, at least about 150 mM, 200 mM, 250mM, 300 mM, 350 mM, 400 mM, 450 mM or 500 mM.

Illustrative reducing agents for use in the solution for solubilizingthe lipid-soluble antigens include without limitation Tributylphosphine(TBP), beta mercaptoethanol, 2-mercaptoethylamine,tris(2-carboxyethyl)phosphine (TCEP), cysteine-HCl, and dithiothreitol(DTT). In some embodiments, the reducing agent is DTT.

Illustrative protease inhibitors for use in the solution forsolubilizing the lipid-soluble antigens include without limitationaspartic protease inhibitors, cysteine protease inhibitors,metalloprotease inhibitors, serine protease inhibitors (serpins),threonine protease inhibitors, trypsin inhibitors, and mixtures thereof.In various embodiments, the protease inhibitor is an 19, 110, 114, 124,129, 134, 136, 142, 148, 153, 167, 168, 178 inhibitor, or a mixturethereof. In some embodiments, the protease inhibitor is AEBSF(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), also sold asPEFABLOC®. In some embodiments, the protease inhibitor isPhenylmethylsulfonyl fluoride (PMSF). Numerous protease inhibitorcocktails of use are commercially available from Roche MolecularBiochemicals.

In some embodiments, the lipid-soluble antigens are solubilized in asolution that comprises one or more of an antibacterial agent and/or anantifungal agent.

In some embodiments, the lipid-soluble antigens are solubilized in asolution that comprises a chelation agent. Illustrative chelation agentsinclude without limitation ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), Citric Acid,N,N-bis(carboxymethyl)glycine (NTA), and the meso isomer ofDimercaptosuccinic acid (DMSA).

In some embodiments, the lipid-soluble antigens are solubilized in asolution comprising Tris-HCl, dithiothreitol (DTT), a proteaseinhibitor, KCl and ASB-14. In a particular embodiment, the lipid-solubleantigens are solubilized in a solution comprising 10 mM Tris-HCl, 100 mMDTT, 100 mM KCl, 2 mM MgCl₂ and 1-4% ASB-14. A protease inhibitor and/oran antibacterial agent and/or an antifungal agent may also be included.

f. Separating the Tissue from the Solubilized Lipid-Soluble Antigens

Once the tissue has been saturated with (e.g., submerged in or perfusedwith) the solution for solubilizing the lipid-soluble antigens for asufficient time, the tissue can be separated from the solution, nowcontaining a portion of the lipid soluble antigens extracted from thetissue. Separation of the tissue and the solubilizing solution can beperformed using any methods known in the art. In various embodiments,the tissue can be rinsed, e.g., in the same solution for solubilizingthe lipid-soluble antigens. Optionally, the tissue can be saturated with(e.g., submerged in or perfused with) and separated for one or moreiterations in fresh solution for solubilizing the lipid-soluble antigensfor a sufficient time to extract a further portion of the lipid solubleantigens from the tissue or to reach an equilibrium between thelipid-soluble antigens within the tissue and lipid soluble antigens inthe solution, as appropriate. The iterations of submerging or perfusingin solution for solubilizing the lipid-soluble antigens and separating(e.g., rinsing) can be repeated until extracted lipid-soluble antigensare no longer detected in the solution for solubilizing thelipid-soluble antigens, or until the detectable extracted lipid-solubleantigens fall below a predetermined threshold level, as appropriate.

In various embodiments, the separation and removal of the lipid-solubleantigens from the tissue can involve centrifugation and/or filtration,as appropriate, to separate the tissue from the solution forsolubilizing the lipid-soluble antigens containing a portion of and/orsaturated with extracted lipid-soluble antigens. In other embodiments,the tissue is removed from solution for solubilizing the lipid-solubleantigens and rinsed with being subjected to centrifugation orfiltration.

In various embodiments, more frequent iterations of submerging orperfusing and separation (e.g., rinsing) and a larger number ofiterations of submerging or perfusing and separating (e.g., rinsing) canallow for faster solubilization and removal of lipid-soluble antigensfrom the tissue. For example, the tissue can be saturated with (e.g.,submerged in or perfused with) a first solution for solubilizing thelipid-soluble antigens for a sufficient time to extract at least aportion of the lipid soluble antigens; separated from the firstsolution, now containing a portion of extracted lipid soluble antigens,before equilibrium is reached between the lipid-soluble antigens withinthe tissue and lipid soluble antigens in the solution; saturated with(e.g., submerged in or perfused with) a second solution for solubilizingthe lipid-soluble antigens for a sufficient time to extract at least aportion of the lipid soluble antigen; and separated from the secondsolution, now containing a portion of extracted lipid soluble antigens,before equilibrium is reached between the lipid-soluble antigens withinthe tissue and lipid soluble antigens in the solution. Furtheriterations of submerging or perfusing and separation can be performed,as appropriate or desired, until a sufficiently low level of lipidsoluble antigens extracted into the solution for solubilizing thelipid-soluble antigens is achieved.

g. Recellularizaton/Repopulation with Live Cells

In various embodiments, the tissue scaffold and/or decellularized ECM(e.g., heart valves, vascular conduits, arteries, veins, skin, dermis,ligaments, tendons, bone, cartilage, muscle, ureter, urinary bladder,liver, heart, and other organs) can be recellularized or repopulatedprior to implantation by co-culturing the tissue processed according toa method of the invention with live cells, e.g., cells autologous to therecipient of the tissue; cells of the same tissue type as the tissue tobe transplanted into the recipient; mesenchymal stem cells; and mixturesthereof.

In various embodiments, the tissue scaffold and/or decellularized ECM isrepopulated or recellularized with mesenchymal stem cells exhibitimmunomodulatory properties through mechanisms involving bothcell-to-cell contact and secretion of soluble factors (PGE2, TGF-β1,IL-6 and hepatocyte growth factor (HGF)). Specifically, in theallogeneic setting, MSCs induce T-cell anergy, reduce dendritic celltype 1 (DC1) TNF-α secretion, increase DC2 IL-10 secretion, decreaseT-helper cell type 1 (Th1) IFN-γ secretion, increase Th2 IL-4 secretion,increase the proportion of regulatory T-cells (Treg) and decreasenatural killer (NK) cell IFN-γ secretion. Furthermore, previous studieshave demonstrated the ability of MSCs to reconstitute all heart valvelayers, making them a potentially ideal source for recellularization ofa tissue engineered heart valve scaffold. Finally, MSCs exhibitmultipotent capacity, autologous availability, clinical relevance andincreased proliferative capacity compared to terminally differentiatedcells. Recellularizaton with MSCs complements antigen removal inproducing an immunologically-acceptable, recellularized tissue scaffold.

As appropriate, in vitro co-culture conditions can, in specificembodiments, be under static conditions for cell culture or can takeplace in a bio-reactor mimicking certain desired in vivo conditions. Asmentioned above, processed tissue can, in some embodiments, be treatedwith growth factors (e.g., basic fibroblast growth factor) or chemokinesto enhance cellular ingrowth/migration into the tissue and/or to directcells to adopt appropriate phenotypes. Such treatments could be employedto enhance in vitro recellularization before implantation of therecellularized tissue scaffold or ECM or to enhance in vivorecellularization after implantation of the tissue scaffold and/ordecellularized ECM, and would be familiar to the ordinarily skilledartisan.

In further embodiments, the tissue scaffolds and/or decellularized ECMcan be treated or impregnated with agents, e.g., growth factors and/orpharmaceuticals. Growth factors may, for example, be used to promoterecellularization, vascularization, or epithelialization. Antibodies orantibiotics may be used to prevent potential infection from implant.Matrix components may also be used. Other so-called recellularizationagents include, without limitation, chemoattractants, cytokines,chemokines, and derivatives thereof.

3. Decellularization and Antigen Removal from Muscle Tissue

In embodiments where the tissue is a muscle tissue, e.g., cardiac muscletissue, striated or skeletal muscle tissue, or smooth muscle tissue, themethods can further comprise the steps of relaxing the muscle tissue andwashing the muscle tissue in a concentrated salt solution to disassembleand remove the sarcomeric components.

Muscle tissue, including cardiac muscle tissue, contains four main celltypes: endothelial cells, fibroblasts, myocytes and smooth muscle cells.Cardiomyocytes have the highest percentage in volume of myocardium.Cardiomyocytes, as well as myocytes in striated or skeletal muscletissue, are made up of sarcomeres aligned in series and in parallel.Since actin filaments represent an important component of the sarcomerestructure, we investigated the addition of an actin depolymerizationstep (e.g., utilizing swinholide, mycalolide B, Latrunculin B and/orCytochalasin D) to convert filamentous cytoskeletal (cortical) andα-sarcomeric actin (F-actin) to globular actin monomers (G-actin).Conversion of F-actin to G-actin monomers increased solubility andtherefore the removal of actin monomers, associated cytoskeletalproteins and antigens. Actin depolymerization alone was insufficient tofacilitate complete solubilization of all macromolecular components ofthe sarcomere. An integrated multimodal approach is employed thatcomplements the actin depolymerization step with additional stepsspecifically targeted to remove sarcomeric myosin and titin. Bathingmuscle tissue in a basic “relaxing solution” designed to render themyosin heads on the sarcomeric apparatus in the relaxed state (e.g.,switched from the ADP-bound or “rigor state” to the ATP-bound or relaxedstate) prior to the antigen removal improved removal of sarcomericproteins and associated antigens from the resulting scaffold. In varyingembodiments, antigen removal and decellularization of muscle tissuecomprises the step of relaxation of the muscle tissue prior to theantigen removal process, followed by removal of lipid-solubleproteins/antigens, sarcomeric depolymerization and removal of specificsarcomeric constituents, and finally removal of water-solubleproteins/antigens. In varying embodiments, antigen removal anddecellularization of muscle tissue comprises the step of relaxation ofthe muscle tissue prior to the antigen removal process, followed bysarcomeric depolymerization and removal of specific sarcomericconstituents, removal of water-soluble proteins/antigens and finallyremoval of lipid-soluble proteins/antigens. This multi-targeted approachresults in successful removal of essentially all detectablemacromolecular sarcomeric components and has a significant impact onremoval of both sarcomere-associated, and non-sarcomere-associatedantigens from the material.

a. Relaxation of Muscle Tissue

The muscle tissue is contacted with (e.g., submerged in or perfusedwith) a relaxing solution comprising an energy source molecule undersufficient conditions and for a sufficient time to render the myocytesarcomere structure (e.g., the functional rigid force-producing unitsthat make up myocytes) amenable to subsequent disassembly andsolubilization. In the relaxation step, a substantial portion of themyosin heads on the sarcomeric apparatus are switched from the ADP-boundor “rigor state” to the ATP-bound or relaxed state. In varyingembodiments, the energy source molecule is selected from the groupconsisting of a nucleotide 5′-triphosphate (NTP), adenosine, inosine,aspartate, glutamate, creatine phosphate, a Kreb's cycle precursor orintermediate, glucose, and dextrose. In some embodiments, the energysource molecule is pyrophosphate (PPi) or a nucleotide 5′-triphosphate(NTP) selected from the group consisting of adenosine 5′-triphosphate(ATP), inosine 5′-triphosphate (ITP), guanidine 5′-triphosphate (GTP),cytidine 5′-triphosphate (CTP), and uridine 5′-triphosphate (UTP). Insome embodiments, the energy source molecule is a precursor of adenosine5′-triphosphate (ATP). In some embodiments, the energy source moleculeis adenosine 5′-triphosphate (ATP). In some embodiments, the energysource molecule is Pyrophosphate (PPi). In some embodiments, the energysource molecule comprises vanadate and adenosine 5′-diphosphate (ADP).

In varying embodiments, the relaxing solution comprises each of the oneor more energy source molecules in a concentration range of about 1 mMto about 200 mM, e.g., about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8mM, 9 mM, 10 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175 mM, or200 mM. In varying embodiments, the relaxing solution comprises 4 mMadenosine 5′-triphosphate (ATP). In varying embodiments, the relaxingsolution comprises 50-200 mM glucose, glutamate and/or aspartate.

In varying embodiments, the relaxing solution is a physiologicallyisotonic aqueous solution. In some embodiments, the relaxing solutionpromotes disassembly of sarcomeric macromolecules as well as thesolubilization of water soluble antigens, as described above in thesection entitled “Solubilizing Water-Soluble Antigens in the Tissue,”further comprising one or more energy source molecules.

In various embodiments, the tissue is submerged in or perfused with therelaxing solution for at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22 or 24 hours, e.g., for at least about 2, 3, 4, 5, 6, 7 days,as appropriate. The tissue may be washed one or more times during thetime period of submerging or perfusing, e.g., to promote disassembly ofsarcomeric components within the muscle tissue.

In various embodiments, relaxation of the muscle tissue is performed ata temperature above freezing (e.g., above 0° C.) and at or below bodytemperature (e.g., at or below about 37° C.). In various embodiments,relaxation of the muscle tissue is performed at a refrigeratedtemperature, e.g., between about 4-10° C. In various embodiments,relaxation of the muscle tissue is performed at room temperature, e.g.,between about 20-30° C., e.g., about 25° C. In various embodiments,relaxation of the muscle tissue is performed at human body temperature,e.g., about 37° C.

In some embodiments, the relaxing solution further comprises a calciumion chelating agent. Illustrative chelation agents include, withoutlimitation, ethylenediaminetetraacetic acid (EDTA),diethylenetriaminepentaacetic acid (DTPA), Citric Acid,N,N-bis(carboxymethyl)glycine (NTA), and the meso isomer ofDimercaptosuccinic acid (DMSA). In some embodiments, the relaxingsolution further comprises a permeabilization agent. In varyingembodiments, the permeabilization is a detergent, e.g, as describedabove and herein. Illustrative detergents for use as permeabilizationagents include without limitation, e.g., Triton-X-100, saponin and/or asulfobetaine (e.g., ASB-14).

In a particular embodiment, the relaxing solution comprises one or morecytoskeletal destabilizing agents in a solution comprising 10 mMTrisHCl, pH 7.6; 0.12 M KCl; 4 mM MgCl₂ 6H₂O; 4 mM EDTA (hydrated) and4-6 mM energy source molecule (e.g., Na-ATP or ATP)). In varyingembodiments, the relaxing solution may further comprise a proteaseinhibitor and one or more antimicrobial agents, e.g., 0.5 mM AEBSF(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride) (“PEFABLOC™”)and 1% Antibiotic Antimycotic Solution (AAS).

b. High Salt Concentration Wash to Facilitate Removal of SarcomericComponents

In varying embodiments, the muscle tissue is exposed to (e.g., submergedin or perfused with) a concentrated salt solution to facilitate thesolubilization and removal of sarcomeric components. In varyingembodiments, the concentrated salt solution comprises one or more saltsin a concentration range from about 0.5 M to about 3.0 M, e.g., about0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M,1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M, 2.3 M, 2.4 M, 2.5 M,2.6 M, 2.7 M, 2.8 M, 2.9 M, or 3.0 M. In some embodiments, theconcentrated salt solution comprises one or more metal halide salts. Insome embodiments, the metal halide salt is selected from the groupconsisting of LiF, LiCl, LiBr, LiI, NaF, NaCl, NaBr, NaI, KF, KCl, KBr,KI, RbF, RbCl, RbBr, RbI, CsF, CsCl, CsBr, CsI, BeF₂, BeCl₂, BeBr₂,BeI₂, MgF₂, MgCl₂, MgBr₂, MgI₂, CaF₂, CaCl₂, CaBr₂, CaI₂, SrF₂, SrCl₂,SrBr₂, SrI₂, BaF₂, BaCl₂, BaBr₂, BaI₂, and mixtures thereof. In someembodiments, the concentrated salt solution comprises KCl and/or KI. Insome embodiments, the tissue is concurrently exposed to KCl and KI, andthe concentrated salt solution comprises 0.6 M KCl and 1.0 M KI. Invarying embodiments, the tissue is sequentially exposed to KCl and KI,e.g., first 0.6 M KCl and then 1.0 M KI or first 1.0 M KI and then 0.6 MKCl. The tissue can be washed in an isotonic solution between exposureto the high concentration salt solutions. As appropriate, the highconcentration salt solution may further comprise a protease inhibitorand one or more antimicrobial agents, e.g., 0.5 mM AEBSF(4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride) (“PEFABLOC™”)and 1% Antibiotic Antimycotic Solution (AAS).

In varying embodiments, the muscle tissue is submerged in or perfusedwith the concentrated salt solution. In some embodiments, the muscletissue is washed or rinsed with the concentrated salt solution. In someembodiments, the relaxing solution, described above, comprises one ormore salts at a high concentration. In varying embodiments, the muscletissue is contacted with the concentrated salt solution after exposureto the relaxing solution.

In some embodiments, sarcomeric constituents are not detectable in themuscle tissue. In some embodiments, at least about 90% of the sarcomericconstituents are removed, e.g., at least about 93%, 95%, 97%, 98%, 99%or all (100%) sarcomeric constituents are removed.

4. Tissue Scaffolds and/or Decellularized Extracellular Matrix (ECM)

Removing antigen components from tissue by sequentially immersing thetissue in separate solutions for depolymerizing and/or destabilizing oneor more cytoskeletal components, solubilizing water soluble antigencomponents and for solubilizing lipid soluble antigen componentsproduces tissue scaffolds and/or decellularized extracellular matrix(ECM) with biophysical and biochemical properties substantially the sameas the tissue before it is subject to antigen removal. The producedtissue scaffolds and/or decellularized extracellular matrix (ECM)moreover have low residual antigenicity (e.g., to an allogeneic orxenogeneic host), and do not elicit significant or destructive immuneresponses by the host against the tissue scaffolds and/or decellularizedextracellular matrix (ECM). The tissue scaffolds and/or decellularizedextracellular matrix (ECM) produced according to the methods describedherein are also non-toxic to live cells and suitable for repopulationand/or recellularization with live cells, e.g., that are allogeneic orxenogeneic to the tissue scaffolds and/or decellularized extracellularmatrix (ECM).

With respect to residual antigenicity, the tissue scaffolds and/ordecellularized extracellular matrix (ECM) that have been subject tosequential solubilization procedures for removal of water-soluble andlipid-soluble antigenic components are substantially depleted of watersoluble and lipid soluble antigen components. In various embodiments, atleast about 80%, for example, at least about 85%, 90%, 93%, 95%, 97%,99%, or more, of the water soluble antigens are removed from the tissueto produce the present tissue scaffolds and/or decellularizedextracellular matrix (ECM) (e.g., the residual antigenicity ofwater-soluble antigens is less than about 20%, e.g, less than about 15%,10%, 7%, 5%, 3%, 1%, or less). In some embodiments, at least about 60%,for example, at least about 65%, 70%, 75%, 80%, 85%, 90%, 93%, 95%, 97%,99%, or more, of the lipid soluble antigens are removed from the tissueto produce the present tissue scaffolds and/or decellularizedextracellular matrix (ECM) (e.g., the residual antigenicity oflipid-soluble antigens is less than about 40%, e.g, less than about 35%,30%, 25%, 20%, 15%, 10%, 7%, 5%, 3%, 1%, or less). In variousembodiments, the water-soluble and/or lipid soluble antigen componentsare not detectable in the produced tissue scaffolds and/ordecellularized extracellular matrix (ECM). The presence and/or amount ofwater-soluble and/or lipid soluble antigen components remaining in thetissue scaffolds and/or decellularized extracellular matrix (ECM) afterperforming antigen removal can be detected using methods known in theart, e.g., in vitro and/or in vivo immunoassays, Western blotting,ELISA, gel electrophoresis, lymphocyte proliferation and/or migrationassays, etc.

For determination of residual antigenicity in vivo, tissue scaffoldsand/or decellularized ECM produced according to the methods describedherein (e.g., repopulated or not repopulated with live cells) can beimplanted subcutaneously in a host that is allogeneic or xenogeneic tothe tissue scaffold or decellularized ECM. Post-implantation developmentof scaffold calcification, innate (histologic assessment), humoral(biomaterial specific IgG positivity, C4d deposition) and cell-mediated(IHC for CD4/CD8/Treg subtyping, lymphocyte proliferation and migrationassays) immune responses can be assessed and compared to both positive(native tissue) and clinically relevant negative control tissue (e.g.,native allograft heart muscle tissue/patch, glutaraldehyde-fixedtissue). Such assays are known in the art. In various embodiments, theproduced tissue scaffolds and/or decellularized extracellular matrix(ECM) do not elicit a detectable immune response, e.g., by lymphocytesallogeneic or xenogeneic to the tissue scaffold and/or ECM as measuredin in vitro and/or in vivo assays. In various embodiments, the producedtissue scaffolds and/or decellularized extracellular matrix (ECM) elicita detectable but insignificant and/or non-destructive immune response,e.g., by lymphocytes allogeneic or xenogeneic to the tissue scaffoldand/or ECM as measured in in vitro or in vivo assays. In variousembodiments, the produced tissue scaffolds and/or decellularizedextracellular matrix (ECM) have a residual antigenicity and elicit animmune response that is equal to or less than the immune response of anegative control tissue (e.g., native allograft heart muscletissue/patch, glutaraldehyde-fixed tissue).

With respect to biophysical properties, the tissue scaffolds and/ordecellularized extracellular matrix (ECM) produced according to thepresent methods retain structure and strength that are not significantlydifferent from or are substantially the same as the tissue before it issubject to antigen removal, e.g., by sequential solubilization ofwater-soluble and lipid-soluble antigen components. For example, thematrix morphology or structural integrity of the tissue scaffolds and/ordecellularized extracellular matrix (ECM) is substantially intact—notsignificantly collapsed, degraded, shrunken, buckled, twisted orotherwise deformed. The size, e.g., length, width, thickness and/orvolume, of the tissue scaffolds and/or decellularized extracellularmatrix (ECM) are not significantly different from or are substantiallythe same as the tissue before it is subject to antigen removal. Thestructure of the tissue scaffolds and/or decellularized ECM producedaccording to the present methods retain a size, shape and structuralintegrity that is not significantly different from or is substantiallythe same as the tissue before it is subject to antigen removal. Thestructural integrity of the produced tissue scaffolds and/ordecellularized ECM can be determined using any method in the art, e.g.,including histology and electron microscopy (e.g., transition electronand/or scanning electron microscopy). See, e.g., Williams, et al., ActaBiomater. (2009) 5(4):993-1005; Zou and Zhang, J Surg Res. (2011) PMID21571306. Histology techniques can be utilized to assess tissuemorphology, residual nuclei counts (e.g., Hematoxylin and Eosin (H&E)staining), and gross ECM structure (e.g., Verhoeff-van Gieson staining)Collagen fiber orientation, mean fiber diameter and percent volume canbe evaluated using electron microscopy. The tissue scaffolds and/ordecellularized extracellular matrix produced according to the presentmethods moreover retain tensile properties, e.g., as measured by Young'smodulus, tensile stress, and tensile strain parameters, that are notsignificantly different from or are substantially the same as the tissuebefore it is subject to antigen removal. Methods for determiningbiophysical properties of a tissue, before and after antigen removal,are known in the art and find use. For example, the size and/or volumeof a sample of tissue can be determined before and after antigenremoval, e.g., using a calipers. Methods for determining Young'smodulus, tensile stress, and/or tensile strain of a tissue are alsoknown in the art and find use. Illustrative methods are described, e.g.,in Wong, et al, Biomaterials. (2011) 32:8129-8138; Ling Y. AMP J. Tech.(1996) 5:37-48; Sheridan, et al., J Mech Behav Biomed Mater. (2012)8:58-70; Williams, et al., Acta Biomater. (2009) 5(4):993-1005; Zou andZhang, J Surg Res. (2011) PMID 21571306; and Petersen, et al., CellsTissues Organs. (2012) 195(3):222-31.

With respect to biochemical properties, the tissue scaffolds and/ordecellularized extracellular matrix (ECM) produced according to thepresent methods retain quantitative biochemical properties that are notsignificantly different from or are substantially the same as the tissuebefore it is subject to antigen removal, e.g., by sequentialsolubilization of water-soluble and lipid-soluble antigen components.For example, quantitative content (e.g., amounts and/or ratios) ofwater, elastin, collagen, glycosaminoglycans and/or proteoglycans is notsignificantly different or is substantially the same in tissues beforeand after antigen removal by the present methods. Methods forquantitative determination of biochemical properties of a tissue sample,e.g., content of water, elastin, collagen, glycosaminoglycans (GAG),proteoglycans, and/or other ECM components, are known in the art andfind use. Illustrative methods are described, e.g., in Wong, et al,Biomaterials. (2011) 32:8129-8138; Williams, et al., Acta Biomater.(2009) 5(4):993-1005; Petersen, et al., Cells Tissues Organs. (2012)195(3):222-31 and Woessner. Arch Biochem Biophys (1961) 93:440e7. Assaykits for determining the content of collagen, elastin, proteoglycansand/or GAG find use and are commercially available, e.g., from BiocolorLtd. (on the internet at biocolor.co.uk), Worthington Biochemical Corp.(on the internet at worthington-biochem.com), Sigma-Aldrich (on theinternet at sigmaaldrich.com), Quickzyme (on the internet atquickzyme.com), Kamiya Biomedical Company (on the internet atkamiyabiomedical.com) and Astarte Biologics (on the internet atastartebio.com).

With respect to live cell repopulation and/or recellularization, thetissue scaffolds and/or decellularized extracellular matrix (ECM)produced according to the present methods are suitable for repopulationor recellularization with live cells. Generally, the tissue scaffoldsand/or decellularized ECM do not contain toxic contaminants or residuefrom the antigen removal process that are toxic to live cells and impedethe ability of live cells to repopulate or recellularize the tissuescaffold or ECM. For example, in various embodiments, the tissuessubject to sequential solubilization of water-soluble and lipid-solubleantigens for antigen removal are not contacted with sodium dodecylsulfate (SDS). In various embodiments, the tissue scaffolds and/ordecellularized extracellular matrix (ECM) produced according to thepresent methods are substantially repopulated with live cells, e.g., atleast about 60%, 65%, 70%, 75%. 80%, 85%, 90%, 95%, 99%, or more, ofcapacity repopulated or recellularized. The extent of recellularizationcan be determined using any method in the art. Illustrative assays forassessing recellularizaton capacity include without limitation toxicity(e.g., LDH), adhesion (e.g., histology), viability (e.g., Live/Dead),migration (e.g., hMSC tissue invasion on histology andimmunohistochemistry (IHC)), and proliferation (e.g., proliferating cellnuclear antigen antibody (PCNA)) assays. As appropriate and depending ontissue to be recellularized and cell/donor availability, the tissuescaffolds and/or decellularized ECM can be repopulated and/orrecellularized with cells autologous to the recipient of the tissue;allogeneic to the recipient of the tissue; cells of the same tissue typeas the tissue to be transplanted into the recipient; mesenchymal stemcells; and mixtures thereof.

Tissue scaffolds and/or decellularized ECM produced according to themethods described herein have application in processing and implantationof bioprostheses, biomaterials, or xenogeneic tissues (xenografts)including, e.g., heart valves, vascular conduits, arteries, veins, skin,dermis, ligaments, tendons, bone, cartilage, muscle, ureter, urinarybladder, liver, heart, and other organs; or processing andtransplantation of fresh, preserved, or banked allogeneic tissues(allografts) including vessels, vascular conduits, arteries, veins heartvalves, skin, dermis ligaments, tendons, bone, cartilage, muscle,ureter, urinary bladder, liver, heart, or other organs; or thedevelopment of natural biological matrices for tissue-engineered tissuesand organs including heart valves, vessels, skin, dermis, ligaments,bone, cartilage, muscle, ureter, urinary bladder, liver, heart, or otherorgans.

In various embodiments, the tissue scaffolds and/or decellularized ECMcan be used as implants, tissue fillers, burn dressings, wounddressings, blood vessel grafts, blood vessel replacements, and the like.Medical graft materials comprising the tissue scaffolds and/ordecellularized ECM produced by sequential antigen removal methods can beused in the repair or reconstruction of tissues such as nervous tissue,dermal tissue (ex: in wound care), cardiovascular tissue (includingvascular and cardiac), pericardial tissue, muscle tissue, bladdertissue, ocular tissue, periodontal tissue, bone, connective tissue(tendons, ligaments), and the like. Medical graft materials of theinvention can also be used in conjunction with one or more secondarycomponents to construct a medical device (e.g., a balloon-expandable orself-expanding stent).

Another application of a method according to the invention is tomitigate, reduce, inhibit and/or prevent immune rejection oftransplanted tissues/organs between individuals within a species (i.e.,allografts) or between species (i.e., xenografts) by the removal ofantigens from the tissue/organ.

If the tissue scaffold and/or decellularized ECM is sterilized followingthe antigen removal procedure, it should have a shelf life of at least 1year, or more.

5. Kits

The invention further provides kits comprising tissue scaffolds and/ordecellularized ECM, e.g., as described herein and/or produced accordingto the methods described herein. The kits may further compriseinstructions for repopulation or recellularization of the tissuescaffolds and/or decellularized ECM with live cells. Generally, tissuescaffolds and/or decellularized ECM provided in such kits are sterilizedand ready for transplantation into a host.

Also provided are kits for producing tissue scaffolds and/ordecellularized ECM substantially depleted of antigenic components andsuitable for live cell repopulation or recellularization. In someembodiment, the kits comprise a first container comprising a solutionfor solubilizing water-soluble antigens and a second containercomprising a solution for solubilizing lipid-soluble antigens. Invarying embodiments, the kits comprise a first container comprising asolution for depolymerizing one or more cytoskeletal components, asecond container comprising a solution for solubilizing water-solubleantigens and a third container comprising a solution for solubilizinglipid-soluble antigens. The embodiments of the solutions fordepolymerizing one or more cytoskeletal components, for solubilizingwater-soluble antigens and for solubilizing lipid-soluble antigens aredescribed above and herein. In varying embodiments, the kits may furthercomprise a container comprising a relaxing solution and a containercomprising a concentrated salt solution. The embodiments of the relaxingsolution and the concentrated salt solution are described above andherein. The kits may further comprise instructions for removingantigenic components from a tissue, e.g., a muscle tissue. In variousembodiments, the kits also comprise a control tissue scaffold and/ordecellularized ECM, e.g., that has been processed employing sequentialantigen removal and produced according to methods described herein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Stepwise Solubilization-Based Antigen Removal for XenogeneicScaffold Generation in Tissue Engineering

Materials and Methods

Tissue harvest. All chemicals were from Sigma-Aldrich (St. Louis, Mo.)unless otherwise stated. Fresh BP was harvested from adult cattle aspreviously described [Wong, et al., Biomaterials (2011) 32:8129-38](n=3).

Antiserum Production.

All animal procedures were conducted in accordance with the guidelinesestablished by University of California, Davis IACUC and the Guide forthe Care and Use of Laboratory Animals [National Research CouncilInstitute of Laboratory Animal Resources Commission on Life Sciences.Guide for the Care and Use of Laboratory Animals. Washington, D.C.:National Academy Press; 1996]. Anti-native BP serum was generated fromNew Zealand white rabbits (n=4) as previously described [Griffiths, etal., Biomaterials (2008) 29:3514-20; Wong, et al., Biomaterials (2011)32:8129-38; Griffiths, et al., Electrophoresis (2008) 29:4508-15].Briefly, following subcutaneous injection of BP homogenate and Freund'sadjuvant at a 1:1 ratio into New Zealand white rabbits (n=4) on days 0,14 and 28, blood was collected at day 84. Serum was isolated followingcentrifugation at 3000 rpm for 10 min and stored at −80° C. until usedon Western blots (Section 2.4).

Protein Extraction.

Protein extraction from minced BP-AR was adapted from a method describedpreviously [Wong, et al., Biomaterials (2011) 32:8129-38; Griffiths, etal., Electrophoresis (2008) 29:4508-15]. All centrifugation steps wereperformed at 17,000 g, 4° C. for 25 min. Briefly, minced BP-AR wasincubated in standard extraction solution (10 mM Tris-HCl (pH 8.0)containing 1 mM dithiothreitol, 2 mM magnesium chloride hexahydrate, 10mM potassium chloride and 0.5 mM Pefabloc SC (Roche, Indianapolis,Ind.)) containing 0.1% (w/v) SDS (Bio-Rad, Hercules, Calif.) at 1000rpm, 4° C. for 1 h. Following centrifugation, recovered supernatant wasdefined as residual hydrophilic protein extract. The insoluble pelletwas washed twice in standard extraction solution containing 0.1% (w/v)SDS at 1400 rpm, 4° C. for 30 min and then incubated in standardextraction solution containing 1% (w/v) SDS at 1400 rpm, 4° C. for 1 h.Following centrifugation, recovered supernatant was defined as residuallipophilic protein extract. All extracts were stored at −80° C.

One-Dimensional Electrophoresis and Western Blot.

One-dimensional electrophoresis and Western blot was performed aspreviously described [Wong, et al., Biomaterials (2011) 32:8129-38],using equal volumes of residual hydrophilic or lipophilic proteinextract per group.

Antigen Removal.

Antigen removal was adapted from a method previously described [Wong, etal., Biomaterials (2011) 32:8129-38]. All steps were performed in a 2 mlworking volume at 4° C. and 125 rpm unless otherwise stated. Briefly,intact pieces of BP (0.2 g, approximately 1.0 cm×1.5 cm) were subjectedto hydrophile solubilization for 2 days as the first step of AR. Thiswas followed by lipophile solubilization at room temperature for 2 daysas the second step of AR. For each AR sample, an anatomically adjacentpiece of BP subjected to AR for 1 min served as a negative AR controlfor biological tissue variability and effects of AR additives. Followingnucleic acid digestion for 24 h and washout for 48 h, BP-AR was storedin Dulbecco's modified Eagle's medium with 15% (v/v) dimethyl sulfoxideat −80° C. All AR experiments were conducted with n=6 per group.

Effect of Hydrophile Solubilization (One-Step AR).

Residual lipophilic antigenicity of BP-AR was assessed after hydrophilesolubilization with either basic AR buffer (BARB; 10 mM Tris-HCl (pH8.0) containing 0.5 mM Pefabloc and 1% (v/v) antibiotic antimycoticsolution) or optimized solubilizing AR buffer (opt SARB; BARB containing100 mM dithiothreitol, 2 mM magnesium chloride hexahydrate and 100 mMpotassium chloride) containing: no additional additive, 134 mM3-(benzyldimethylammonio) propanesulfonate (NDSB-256) or 0.1% (w/v) SDS.

Effect of Sequential Hydrophile and Lipophile Solubilization (Two-StepAR).

Both residual hydrophilic and lipophilic antigenicity of BP-AR wereassessed after two-step AR (FIG. 1). Pieces of BP underwent hydrophilesolubilization with opt SARB, followed by lipophile solubilization inopt SARB containing: no additional additive; 134 mM NDSB-256 and 1%(w/v) n-dodecyl-β,D-maltoside (Griffiths solution) [Griffiths, et al.,Biomaterials (2008) 29:3514-20; Griffiths, et al., Electrophoresis(2008) 29:4508-15]; 8 M urea (Bio-Rad), 2 M thiourea, 2% (w/v)3-[(3-cho-lamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 2%(w/v) N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10) and 1%(w/v) 3-[N,N-Dimethyl(3-myristoylaminopro-pyl)ammonio]propanesulfonate(ASB-14) (Cordwell solution) [Cordwell, Methods Mol Biol (2008)424:139-46]; or 10% (v/v) isopropanol (Thermo Fisher Scientific) and 5%(v/v) glycerol (Leimgruber solution) [Leimgruber R M. “Extraction andsolubilization of proteins for proteomic studies,” In: Walker J M,editor. The Proteomics Protocols Handbook. Totowa: Humana Press; (2005)pp. 1-18]. These samples were compared to a literature control of BARBcontaining 0.1% (w/v) SDS for hydrophile solubilization, followed by 1%(w/v) SDS for lipophile solubilization [Griffiths, et al.,Electrophoresis (2008) 29:4508-15].

Effect of Cordwell Solution Components.

Residual lipophilic antigenicity of BP-AR was assessed after hydrophilesolubilization with opt SARB, followed by lipophile solubilization inopt SARB containing: no additional additive; 8 M urea and 2 M thiourea;2% (w/v) CHAPS; 2% (w/v) SB 3-10; 1% (w/v) ASB-14; or the entireCordwell solution during the second step of AR.

Assessment of residual antigenicity following AR. Assessment of residualBP-AR antigenicity was performed using a method previously validated[Griffiths, et al., Biomaterials (2008) 29:3514-20] and described [Wong,et al., Biomaterials (2011) 32:8129-38] on residual hydrophilic orlipophilic protein extracts (FIG. 1). Briefly, residual hydrophilic orlipophilic protein extracts (n=6 per group) were subjected toelectrophoresis and Western blot, probed with anti-native BP serum andassessed for IgG positivity, with band intensity quantified bydensitometry. Residual antigenicity of BP-AR was defined as the ratio ofthe banding intensity from 2 days of AR to that of the 1 min AR control.Residual antigenicity ratios for each AR treatment were then normalizedto the negative AR control within each experiment (BARB for one-step ARor opt SARB alone for two-step AR).

Uniaxial Tensile Testing.

Tensile properties of BP were determined as previously described [Wong,et al., Biomaterials (2011) 32:8129-38]. Strips of BP (15×3 mm) were cutfrom separate 0.2 g pieces of native BP and BP-AR (n=6 per group) andmade into dogbone shapes. Samples from adjacent anatomical locationswere used for each replicate of AR treatment and control tissue. BPmounted under zero strain was subjected to a constant strain rate of 0.1mm s−1. The initial gauge length was set at 2 mm. The initial gaugewidth and thickness were determined from BP images using ImageJ 1.42qsoftware (Wayne Rasband, National Institutes of Health, USA). For eachsample, a stress—strain curve was generated from the load—elongationcurve, and the Young's modulus and ultimate tensile stress (UTS) weredetermined.

Quantitative Biochemistry.

The ECM composition of native BP and BP-AR was determined as previouslydescribed [Wong, et al., Biomaterials (2011) 32:8129-38] from two 5 mmdisks (taken from the initial 0.2 g piece, n=6 per group). One disk wassubjected to papain digestion for quantification of collagen content perdry weight (DW) using a modified colorimetric hydroxyproline assay[Woessner, Arch Biochem Biophys (1961) 93:440-7] and sulfatedglycosaminoglycan (GAG) content per DW using the Blyscan sulfated GAGassay (Biocolor Ltd., Carrickfergus, UK). The second disk was subjectedto hot oxalic acid extraction for quantification of elastin content perDW using the Fastin elastin assay (Biocolor Ltd.).

Histology.

Histological assessment of native BP and BP-AR was performed aspreviously described [Wong, et al., Biomaterials (2011) 32:8129-38] fromtwo 1 mm wide strips (taken from the initial 0.2 g piece, n=6 pergroup). Formalin-fixed, paraffin-embedded sections underwent Verhoeffvan Gieson staining (VVG) for assessment of gross collagen and elastinorganization and hematoxylin and eosin (H&E) staining Assessment of ECMfiber morphology was performed for all six replicates per AR treatmentthroughout the full thickness of the tissue (from the parietal surfaceto the mediastinal surface of the pericardium). Quantification ofresidual nuclei was performed on six randomized high-powered fields(HPFs) per slide. Correlation between nuclei counts and residuallipophilic antigenicity was determined by plotting average nuclei countsagainst residual lipophilic antigenicity for each AR method.

Immunohistochemistry.

Paraffin-embedded sections of native BP and BP-AR samples (n=6 pergroup) were deparaffinized in xylene (Fisher Scientific, Pittsburgh,Pa.) and rehydrated through an ethanol gradient. Endogenous peroxidasewas quenched through incubation in per-oxidase block (Dako, Carpinteria,Calif., USA) for 5 min, followed by antigen retrieval using proteinase K(Dako) for 10 min. Slides were blocked in 5% normal goat serum (NGS)(Jackson ImmunoResearch Laboratories, Inc.) for 30 min and thenincubated in primary antibody for 60 min. Following incubation inEnVision+ anti-mouse HRP-labelled polymer (Dako) for 30 min, boundprimary antibodies were detected using EnVision+ DAB+ chromogen (Dako)for 10 min. Finally, slides were counterstained with Mayer's hematoxylinfor 5 min, dehydrated through an ethanol gradient, cleared in xylene,mounted in Permount (Fisher Scientific) and coverslipped. The twoprimary antibodies used were anti-galactose-a(1,3)-galactose (a-gal)clone M86 (Enzo Life Sciences, Plymouth Meeting, Pa.) at a 1:5 dilutionin 5% NGS and anti-major histocompatibility complex I (MHC I) heavychain (Abcam, Cambridge, Mass.) at a 1:250 dilution in 5% NGS. Sectionsfrom all six replicates per AR treatment were assessed for the presenceof α-gal and MHC I antigens throughout the full thickness of the tissue(from the parietal surface to the mediastinal surface of thepericardium).

Statistical Analysis.

Normalized residual antigenicity ratios were compared betweenexperimental AR groups and the negative solubilization control (BARB forone-step AR and opt SARB alone for two-step AR). Values determined fromtensile testing, biochemical assays and histology were compared to thosefor control tissues (native BP). Non-repeated measures analysis ofvariance and Tukey-Kramer HSD post hoc analysis were performed on samplemeans. Correlation was determined using bivariate fit analysis. All dataare presented as mean±standard deviation from the mean. Statisticalsignificance was defined at p<0.05.

Results

Antigen Removal

Effect of Hydrophile Solubilization (One-Step AR).

Promotion of hydrophile solubilization with opt SARB did notsignificantly change the residual lipophilic antigenicity of BP-ARcompared to BARB for any of the additives assessed: no additionaladditive (0.86±0.14 vs. 1.00±0.51), 134 mM NDSB-256 (0.51±0.12 vs.0.58±0.24) or 0.1% (w/v) SDS (0.35±0.12 vs. 0.42±0.03) (FIG. 2).

Effect of Sequential Hydrophile and Lipophile Solubilization (Two-StepAR).

Promotion of lipophile solubilization during a second step of AR withGriffiths, Cordwell or Leimgruber solution did not significantly changethe residual hydrophilic antigenicity of BP-AR compared to opt SARBalone (0.79±0.28, 0.13±0.11, 0.72±0.27, 1.00±0.15, respectively) (FIG.3A). However, use of opt SARB during the first of two AR steps reducedresidual hydrophilic antigenicity significantly—by 75%—compared to thatremaining following two-step AR with 0.1 and 1% (w/v) SDS in BARB(4.03±2.27, p<0.0005).

Promotion of lipophile solubilization during a second step of ARsignificantly decreased residual lipophilic antigenicity of BP-AR withrespect to that treated with opt SARB alone (1.00±0.22) (FIG. 3B).Compared to the use of opt SARB alone, the percent reduction of residuallipophile antigenicity achieved was 44% with the Griffiths solution(0.66±0.14, p<0.05), 91% with the Cordwell solution (0.09±0.04,p<0.0001) and 33% with the Leimgruber solution (0.67±0.11, p<0.05). Useof 1% (w/v) SDS in BARB resulted in a 37% decrease in residuallipophilic antigenicity (0.63±0.15, p<0.01) compared to opt SARB alone.Furthermore, use of the Cordwell solution in opt SARB during the secondstep of AR significantly reduced residual lipophilic antigenicity by 54%compared to with 1% (w/v) SDS in BARB (p<0.0001).

Effect of Cordwell Solution Components.

Promotion of lipophile solubilization during a second step of AR in optSARB reduced the residual lipophilic antigenicity of BP-AR compared tothat treated with no additional additive (1.00±0.19, p<0.0001)significantly—by 85% with 8 M urea and 2 M thiourea (0.15±0.11), 60%with 1% (w/v) ASB-14 (0.40±0.12) and 94% with the entire Cordwellsolution (0.06±0.02) (FIG. 4A). However, treatment with 1% (w/v) ASB-14in opt SARB did not reduce the residual lipophilic antigenicity of BP-ARto the level achieved with the entire Cordwell solution (p<0.05). Theresidual lipophilic antigenicity of BP-AR treated with opt SARBcontaining 8 M urea and 2 M thiourea was not significantly differentfrom that achieved using either 1% (w/v) ASB-14 or the entire Cordwellsolution.

Gross Tissue Morphology

Effect of Hydrophile Solubilization (One-Step AR).

No change in BP-AR thickness was observed following AR with opt SARB(containing no additional additive, 134 mM NDSB-256, or 0.1% (w/v) SDS)compared to BP-AR generated using BARB, 1 min AR controls or native BP.

Effect of Sequential Hydrophile and Lipophile Solubilization (Two-StepAR).

Treatment with either 1% (w/v) SDS in BARB or the Cordwell solutionduring the second step of AR resulted in gross morphological thickeningof BP-AR compared to that generated using opt SARB alone (FIG. 3C).Moreover, BP-AR subjected to the Cordwell solution curled upward, ratherthan remaining flat. No significant change in BP-AR thickness wasobserved following treatment with the Griffiths or Leimgruber solution.

Effect of Cordwell Solution Components.

Treatment with either 8 M urea and 2 M thiourea in opt SARB or theentire Cordwell solution during the second step of AR resulted in grossmorphological thickening and curling of BP-AR compared to that generatedusing opt SARB alone (FIG. 4B). No significant change in BP-AR thicknesswas observed following treatment with opt SARB containing 2% (w/v)CHAPS, 2% (w/v) SB 3-10 or 1% (w/v) ASB-14.

Uniaxial Tensile Testing.

No significant differences in Young's modulus and UTS were observedbetween native BP (14.25±6.87 and 8.25±2.64 MPa, respectively) and BP-ARgenerated with opt SARB alone (16.43±7.01 and 9.86±2.14 MPa,respectively), 1% (w/v) ASB-14 in opt SARB (11.36±3.94 and 7.52±2.22MPa, respectively) or 1% (w/v) SDS in BARB (8.87±4.23 and 5.48±2.26 MPa,respectively) (FIG. 5). Use of 8 M urea and 2 M thiourea in opt SARBsignificantly decreased the Young's modulus and UTS (1.35±0.55 and1.70±0.68 MPa, respectively) of BP-AR compared to native BP.

Quantitative Biochemistry.

The water content of BP-AR generated using opt SARB containing noadditional additive (74.36±3.67%) or 1% (w/v) ASB-14 (78.74±1.39%)during the second step of AR was not significantly different from thatof native BP (74.73±2.42%) (FIG. 6A). However, use of 8 M urea and 2 Mthiourea in opt SARB (83.43±0.59) or 1% (w/v) SDS in BARB (82.61±2.58%)significantly increased the water content of BP-AR compared to that ofnative BP (p<0.0001).

The collagen content of BP-AR was not significantly different than thatof native BP (34.98±14.75% per DW) following any of the tested lipophilesolubilization treatments: opt SARB alone (48.10±21.07% per dry weight(DW)), 1% (w/v) ASB-14 in opt SARB (55.40±28.60% per DW), 8 M urea and 2M thiourea in opt SARB (31.56±3.99% per DW) or 1% (w/v) SDS in BARB(28.75±9.73% per DW) (FIG. 6B).

The elastin content of BP-AR generated using opt SARB containing noadditional additive (2.58±1.04% per DW) or 1% (w/v) ASB-14 (2.16±1.02%per DW) during the second step of AR was not significantly differentfrom that of native BP (3.09±0.56% per DW) (FIG. 6C). However, use of 8M urea and 2 M thiourea in opt SARB (0.70±0.27% per DW) significantlydecreased the elastin content of BP-AR compared to that of native BP(p<0.0005). The elastin content of BP-AR generated using 1% (w/v) SDS inBARB was also significantly reduced, to a level below the limit ofdetection of the assay (p<0.0001).

The GAG content of BP-AR was significantly different from that of nativeBP (0.75±0.05% per DW) following treatment with opt SARB containing: noadditional additive (0.52±0.09% per DW), 1% (w/v) ASB-14 (0.25±0.10% perDW) or 8 M urea and 2 M thiourea (0.44±0.08% per DW) during the secondstep of AR (p<0.0005) (FIG. 6D). The presence of residual SDS in BP-ARsubjected to 1% (w/v) SDS in BARB during the second step of ARinter-fered with the Blyscan assay.

Histology.

Qualitatively, no differences in collagen and elastin content andorganization were observed between VVG-stained sections of native BP andBP-AR following a second step of AR in opt SARB containing no additionaladditive or 1% (w/v) ASB-14 (FIG. 7). However, BP-AR generated with 8 Murea and 2 M urea in opt SARB or 1% (w/v) SDS in BARB exhibited a markedloss of collagen fiber organization and elastin content. Minordifferences in staining intensity were attributed to processingartifacts and not a change in collagen and elastin organization.

In H&E-stained BP sections, ECM morphology was grossly maintainedfollowing a second step of AR using no additive or 1% (w/v) ASB-14 inopt SARB compared to native BP (FIG. 7). However, treatment with 8 Murea and 2 M thiourea in opt SARB or 1% (w/v) SDS in BARB resulted inmarked disruption of native ECM morphology.

All two-step AR treatments significantly reduced the number of nucleiper HPF compared to native BP (107.42±21.95) (p<0.0001) (FIG. 8). Use ofopt SARB containing no additional additive (14.81±23.44) or 1% (w/v)ASB-14 (3.81±6.47), or 1% (w/v) SDS in BARB (0.75±1.11), significantlyreduced residual nuclei per HPF in BP-AR compared to 8 M urea and 2 Mthiourea in opt SARB (67.56±27.27) (p<0.0001). Moreover, two-step ARusing 1% (w/v) ASB-14 in opt SARB reduced residual nuclei per HPF to asimilar degree to 1% (w/v) SDS in BARB. No statistically significantcorrelation was observed between residual nuclei counts per HPF and thenormalized residual lipophilic antigenicity ratio of BP-AR followingtwo-step AR (p=0.2740, R2=0.5270).

Immunohistochemistry.

Immunohistochemical staining revealed the presence of α-gal and MHC Iantigens in native BP and BP-AR using opt SARB alone (FIG. 7). Lowlevels of α-gal and MHC I antigens localized to vascular structures wereobserved in BP-AR generated with 8 M urea and 2 M thiourea in opt SARB.Although no α-gal antigens were detected in BP-AR using 1% (w/v) SDS inBARB, a low level of MHC I antigens was observed. No detectable α-galand MHC I antigens were found in BP-AR treated with 1% (w/v) ASB-14 inopt SARB or the negative primary antibody controls.

Discussion

There were four objectives to this study: (i) to determine ifsolubilization of one protein subset affects the residual antigenicityof a second protein subset (i.e., is residual lipophilic antigenicityreduced with hydrophile solubilization, or is residual hydrophilicantigenicity reduced with lipophile solubilization?); (ii) to determinewhether a two-step sequential, differential protein solubilization ARstrategy (hydrophile solubilization, followed by lipophilesolubilization) reduces xenogeneic tissue antigenicity beyond thatachieved by a one-step AR method (hydrophile solubilization alone) orthe positive literature control (decellularization with 1% (w/v) SDS);(iii) to identify which of the tested lipophile solubilizing factorsmost effectively reduce residual lipophilic antigenicity of BP-AR in atwo-step AR strategy; and (iv) to assess whether two-step sequential,differential solubilization-based AR methods adversely affectbiomaterial structure-function properties, defined as uniaxial tensileproperties and ECM structure and composition. We demonstrate that: (i)promotion of hydrophile or lipophile solubilization does notsignificantly alter residual lipophilic or hydrophilic antigenicity,respectively; (ii) promotion of hydrophile solubilization, followed bylipophile solubilization, in a two-step sequential, differential ARprocedure enhances the removal of antigens from intact BP beyond thatachieved using a one-step AR method; and (iii) 1% (w/v) ASB-14 enhancesthe removal of lipophilic antigens from BP, eliminating the two mostcritical known barriers to xenotransplantation (a-gal and MHC I),without compromising biomaterial structure-function properties.

Previously, we reported that hydrophile solubilization using a reducingagent and salt (opt SARB) enhances removal of hydrophilic antigens fromBP [Wong, et al., Biomaterials (2011) 32:8129-38]. In the current study,we demonstrate that hydrophile solubilization has no effect on theremoval of lipophilic antigens from BP. Additionally, we show thatapplication of lipophile solubilization as a second AR step has noeffect in further reducing residual hydrophilic antigenicity of BPfollowing one-step AR. Furthermore, the 75% reduction in residualhydrophilic antigenicity observed with opt SARB compared to 1% (w/v) SDSis comparable to our previously published results for residualhydrophilic antigenicity observed with opt SARB compared to 0.1% (w/v)SDS [Wong, et al., Biomaterials (2011) 32:8129-38]. This suggests thatthe increase in concentration of SDS from 0.1% (w/v) to 1% (w/v) doesnot remove markedly more hydrophilic antigens. In sum, these findingsare in agreement with the observation that protein extraction fromhomogenized tissue can only occur into a solution in which theparticular protein subset of interest is soluble [Cordwell, Methods MolBiol (2008) 424:139-46]. Therefore, removal of antigenic proteins fromintact tissue is heavily dependent on the ability of the AR buffer toeffectively solubilize the protein antigen subset(s) of interest.

Persistence of lipophilic antigens following one-step AR underscores theneed for lipophile solubilization in a sequential AR strategy.Incorporation of lipophile solubilizing factors into a second AR stepfacilitates a significant reduction in the residual lipophilicantigenicity of BP-AR compared to hydrophile solubilization alone (optSARB alone). Additionally, lipophile solubilization reduces residuallipophilic antigenicity of BP-AR to a degree comparable to (Griffiths[Griffiths, et al., Biomaterials (2008) 29:3514-20; Griffiths, et al.,Electrophoresis (2008) 29:4508-15] or Leimgruber [Leimgruber R M.“Extraction and solubilization of proteins for proteomic studies,” In:Walker J M, editor. The Proteomics Protocols Handbook. Totowa: HumanaPress; (2005) pp. 1-18] solutions) or beyond (Cordwell solution[Cordwell, Methods Mol Biol (2008) 424:139-46]) that achieved by thecurrent most commonly used decellularization agent (1% (w/v) SDS) [Zhou,et al., Biomaterials (2010) 31:2549-54; Cebotari, et al., Artif Organs(2010) 34:206-10; Gilbert, et al., Biomaterials (2006) 27:3675-83].Furthermore, use of opt SARB for hydrophile solubilization significantlyreduces residual hydrophilic antigenicity compared to 1% (w/v) SDS.Consequently, a two-step sequential, differential AR strategy using optSARB, followed by lipophile solubilization is more effective than 1%(w/v) SDS at reducing both residual hydrophilic and lipophilicantigenicity of BP-AR. Further studies will be necessary to determine ifthe sequence in which solubilization is promoted has any effect on theresidual antigenicity of BP-AR. These findings high-light the importanceof a two-step sequential, differential AR approach, consisting ofhydrophile solubilization followed by lipophile solubilization, foreffective reduction of residual BP antigenicity compared to a one-stepAR methodology or positive decellularization control.

After validating the need for lipophile solubilization, we sought thebest candidate for use in two-step AR. Cordwell solution, the onlytested two-step AR treatment to significantly reduce residual lipophilicantigenicity compared to the literature control (1% (w/v) SDS), reducesthe lipophilic antigenicity of BP-AR impressively—by 91%—compared to optSARB alone, but drastically alters the gross morphological appearance ofBP-AR. Assessment of the individual components of Cordwell solutionrevealed that chaotropes (8 M urea and 2 M thiourea) reduce residuallipophilic antigenicity—by 85%—compared to opt SARB alone. However,chaotropes were also found to be responsible for the gross morphologicaldisruption of BP-AR observed with the entire Cordwell solution. Incontrast, 1% (w/v) ASB-14 reduces residual lipophilic BP-ARantigenicity—by 60%—compared to opt SARB alone, while avoiding thedetrimental changes in gross morphology of BP-AR. Additionally,lipophile solubilization using 1% (w/v) ASB-14 eliminates detection ofthe two most critical known barriers to xenotransplantation (α-gal andMHC I) from resultant BP-AR. The α-gal epitope is a carbohydrate moietypresent on glycolipids and glycoproteins within the cell membrane[Galili, Biochimie (2001) 83:557-63] and the principal determinant ofhyperacute rejection in discordant xenotransplants [Yang, et al., NatRev Immunol (2007) 07:519-31]. The cell surface molecule MHC I is themost ubiquitously known stimulator of both innate and adaptivexenogeneic immune responses [Yang, et al., Nat Rev Immunol (2007)07:519-31; Cascalho, Immunity (2001) 14:437-46]. Thus, removal of theseknown cell membrane-associated xenoantigens is likely to be crucial inreducing recipient immune response to xenogeneic biomaterials.Conversely, persistence of α-gal and MHC I antigens in the absence oflipophile solubilization (opt SARB alone) suggests that BP-AR undergoingonly one-step AR would likely be subject to a substantial immuneresponse. Similarly, detectable α-gal and/or MHC I antigens in BP-ARgenerated with 8 M urea and 2 M thiourea or 1% (w/v) SDS suggest thatthe resultant scaffolds would be unlikely to avoid the immune response.At first glance, these immunohistochemical findings appear to runcounter to the Western blot findings when, in fact, it is likely thatthey are complementary. The rabbit serum used to assess residuallipophilic antigenicity of BP-AR via Western blot comprises polyclonalantibodies to a broad range of antigens, representing the globallipophilic antigenicity of BP-AR. Immunohistochemical analysis of anindividual known antigen using a monoclonal antibody represents asemi-quantitative assessment of residual antigenicity for a specificepitope. Since removal of individual antigens is dependent on theirsolubility in the AR solution used, it is conceivable for a largeproportion of an individual antigen to be removed while globalantigenicity is minimally affected or a small proportion of anindividual antigen be removed while global antigenicity is significantlyreduced. Additional studies will be necessary to determine if thispotential mechanism accurately accounts for the observed discrepancybetween our Western blot and immunohistochemistry results. Furthermore,future in vivo studies will be crucial in determining what level ofxenogeneic scaffold residual antigenicity (including insolublecomponents) is compatible with development of immune tolerance uponimplantation, both for the presented small animal model and ultimatelyin human patients. Nonetheless, the ability of 1% (w/v) ASB-14 tosignificantly reduce residual lipophilic antigenicity and eliminate cellmembrane-associated xenoantigens known to facilitate immune rejection ofxenogeneic tissue makes it a strong candidate for lipophilesolubilization in two-step AR, warranting further characterization ofstructure-function properties for the resultant scaffold.

A successful two-step AR strategy must preserve xenogeneic scaffoldstructure-function properties. Use of 1% (w/v) ASB-14 for lipophilesolubilization resulted in BP-AR with tensile properties, ECMcomposition and ECM organization indistinguishable from native BP. Thisis likely attributed to the ability of zwitterionic detergents such asASB-14 to accomplish lipophile solubilization while maintaining nativeprotein conformation [Nagy, et al., “Sample preparation for 2Delectrophoresis and CE-based proteomics,” Sample preparation inbiological mass spectrometry. New York: Springer; (2011) pp. 41-60]. Thepresence of both hydrophilic and hydrophobic domains on detergentmolecules enables their incorporation into the cell membrane, leading tobilayer destabilization and fragmentation and, ultimately, thesolubilization of resultant detergent-protein complexes [le Maire, etal., Biochim Biophys Acta (2000) 1508:86-111]. In contrast, chaotropessuch as 8 M urea and 2 M thiourea have been reported to achievelipophile solubilization through protein denaturation [Frank, et al., JChem Phys (1968) 48:4746-57; Hua, et al., Proc Natl Acad Sci USA (2008)105:16928-33; Gordon, et al., Biochemistry (1963) 2:47-57].Denaturation, but not loss, of collagen may account for disruptedcollagen fiber organization, and subsequent gross thickening, increasedwater content and altered tensile properties of BP-AR following AR using8 M urea and 2 M thiourea [Hua, et al., Proc Natl Acad Sci USA (2008)105:16928-33; Gordon, et al., Biochemistry (1963) 2:47-57].Additionally, elastin denaturation, manifested as a loss of elastinorganization and content, may also contribute to the unacceptable lossof structure-function properties in chaotrope-treated BP-AR. In aorticvalve leaflets, elastin fibers maintain leaflet structure-functionproperties by facilitating changes in collagen fiber configurationthroughout the cardiac cycle [Vesely, J Biomech (1997) 31:115-23]. Bycompromising elastin-mediated pre-stress on the collagen fibers inBP-AR, 8 M urea and 2 M thiourea may adversely alter collagen fiberorganization and, ultimately, the structure-function properties ofBP-AR. Similarly, lipophile solubilization using 1% (w/v) SDS was alsofound to result in significant alterations to biomaterialstructure-function properties. Increasing concentrations of SDSincreases collagen swelling [Krejc{hacek over ( )}i, Int J Cosmetic Sci(2007) 29:121-9; Henriquez, Macromolecules (1994) 27:6834-40] due todestabilization of the triple helical domain [Samouillan, et al., JBiomed Mater Res (1999) 46:531-8]. Thus, SDS-mediated disruption ofcollagen architecture and removal of elastin fibers may explain thenoticeable gross tissue thickening and increased water content observedin the resultant BP-AR. The structural and compositional alterationsassociated with 1% (w/v) SDS may account for tensile properties trendinglower than those of native BP, although this finding failed to reachstatistical significance. Amongst the antigen removal agents tested fortwo-step AR, 1% (w/v) ASB-14 demonstrates the most promise in xenogeneicscaffold generation by achieving significant reduction in residuallipophilic antigenicity while maintaining structure-function propertiescomparable to native BP. Future studies will be necessary to determinethe compatibility of scaffolds generated using a two-step AR process(opt SARB, followed by 1% (w/v) ASB-14 in opt SARB) withrecellularization, in vivo physiological function and in vivo recipientimmune response.

Previously, we questioned the appropriateness of using residual nucleicounts as the sole indicator of sufficient AR after demonstrating thatoverall residual hydrophilic antigenicity does not correlatesignificantly with residual nuclei counts [Wong, et al., Biomaterials(2011) 32:8129-38]. The lack of significant correlation was notsurprising as one would not expect one-step AR, solely promotinghydrophile solubilization, to efficiently solubilize the nuclearmembrane. Thus, residual nuclei counts were expected to better representresidual lipophilic antigenicity of BP-AR. While lipophilesolubilization reduces residual nuclei counts in BP-AR significantly, nosignificant correlation was found between residual lipophilicantigenicity and residual nuclei counts. As residual nuclei countsmerely serve as an indicator of DNA that persists within the tissue,they do not reflect the level of either residual hydrophilic orlipophilic antigenicity within the biomaterial. Thus, assessment ofbiomaterial decellularization does not provide an accurate assessment ofAR from xenogeneic biomaterials.

Conclusions

By targeting the solubilization of multiple protein subsets using asequential, differential approach (first removing hydrophiles, thenlipophiles), biomaterial antigenicity can be more efficiently reducedcompared to a single solution that only solubilizes one protein antigensubset. Sequential application of opt SARB, followed by 1% (w/v) ASB-14in opt SARB, to BP reduces residual hydrophilic antigenicity by anadditional 75% compared to that achieved by 1% (w/v) SDS in BARB andresidual lipophilic antigenicity by an additional 60% compared to thatachieved by opt SARB alone. Excitingly, this two-step AR methodeliminates the presence of the two most critical known barriers toxenotransplantation (α-gal and MHC I) without significantly compromisingstructure-function properties of the resultant scaffold. In sum, thesefindings illustrate that facilitating the sequential, differentialsolubilization of hydrophiles and lipophiles in a two-step AR strategy,utilizing opt SARB followed by 1% (w/v) ASB-14 in opt SARB, (i)significantly reduces the residual hydrophilic and lipophilicantigenicity of BP— AR, and (ii) maintains biomaterialstructure-function properties. Beyond the generation of BP-derivedscaffolds for heart valve tissue engineering, application of thisstepwise AR strategy to other tissues or organs of the body represents amore efficient alternative to decellularization for the generation ofimmune system-tolerant, tissue engineering scaffolds from xenogeneictissues.

Example 2 In Vivo Implantation of Decellularized Tissue

Results

One week after subpannicular implantation, a non-specific inflammatoryresponse following the surgery was observed toward all bovinepericardium (BP) scaffolds. No dramatic differences in this responsewere observed between treatment groups (FIG. 10).

Six weeks after subpannicular implantation, the in vivo immune responseof New Zealand White rabbits towards BP following stepwise,solubilization-based antigen removal using opt SARB, followed by ASB-14is markedly less than that towards native BP; BP following one-step,solubilization-based antigen removal (opt SARB); BP decellularized with1% (w/v) SDS; and even the current clinically accepted gold standardglutaraldehyde-fixed BP (FIG. 11).

A minimal amount of small mononuclear cells (MNCs) are observed in thesubdermal, subpannicular, and peri-scaffold regions associated withASB-14 treated BP-AR. A mild level of small MNCs is associated withSDS-decellularized BP. A moderate amount of small MNCs were found withfixed BP or native BP. A severe level of small MNCs, including formationof lymphoid follicles, is associated with opt SARB alone-treated BP-AR.Stepwise antigen removal using ASB-14 decreases the amount of MNCs inthe peri-scaffold region compared to native BP, opt SARB-treated BP,SDS-decellularized BP, or fixed BP.

Little to no fibrous encapsulation is seen with ASB-14 treated BP-AR,opt SARB-treated BP-AR, or native BP. Conversely, notable fibrousencapsulation is associated with SDS-decellularized BP or fixed BP.Stepwise antigen removal using ASB-14 is associated with less fibrousencapsulation than SDS-decellularized BP or fixed BP.

Finally, marked cellular ingrowth is observed with ASB-14 treated BP-ARor opt SARB-treated BP-AR. However, minimal cellular ingrowth isassociated with SDS-decellularized BP or fixed BP. Stepwise antigenremoval using ASB-14 supports robust cellular repopulation of BP-AR.

Conclusions

We demonstrate herein that stepwise, solubilization-based antigenremoval using ASB-14 significantly reduces the global residualantigenicity of BP-AR and eliminates the presence of α-gal and MHC I—thetwo most critical barriers to xenotransplantation.

This example provides, for the first time, in vivo observations whichfurther validate our in vitro findings for a significant reduction of BPresidual antigenicity with stepwise, solubilization-based antigenremoval using ASB-14. In contrast to clinically-approved fixed BP orSDS-decellularized BP: (1) Less small MNCs are elicited to respond toASB-14 treated BP-AR at six weeks following implantation. (2) ASB-14treated BP-AR has not been walled off by fibrous encapsulation in therabbit. (3) ASB-14 treated BP-AR is able to support recellularizationwith host cells. Taken together, these findings suggest that stepwise,solubilization-based antigen removal using ASB-14 reduces the in vivoimmune response towards BP-AR compared to native BP; BP followingone-step, solubilization-based antigen removal (opt SARB);SDS-decellularized BP; and fixed BP.

Example 3 Antigen Removal from Cardiac Muscle Tissue

Materials and Methods

Myocardial Patch (MP) Isolation.

Whole hearts were isolated from Fischer rats (CDF®, CRL, Kingston, N.Y.)being euthanized from other non-cardiac studies, perfused with heparinvia antegrade coronary perfusion and stored in storage solution (DMEMwith 15% DMSO v/v) at −80° C. Hearts are defrosted at room temperatureand blood removed by antegrade coronary perfusion with 4° C. heparinizedPBS (10 IU/ml). MPs are prepared as follows: The left ventricle (LV) isisolated and two 4 mm thick short axis LV slices cut from base to apex.Each LV slice is cut longitudinally through the LV free wall between thepapillary muscles. A 3.5 mm biopsy punch (Miltex) is utilized to cutadjacent cylindrical pieces of LV tissue. This approach allows for thegeneration of 6 identical LV myocardial patches (MP) from each LV slice(total of 12 MPs per heart). MPs are placed in Relaxing Solutioncomprising of 120 mM KCl, 4 mM MgCl₂, 4 mM EGTA, 20 mM TrisHCl pH 7.5,5.88 mM NaATP, 0.5 mM Pefabloc, 1% Antibiotic Antimycotic Solution (AAS;Sigma) on ice upon isolation until further processing. MPs are patteddry twice and their wet weights recorded (approximately 20 mg perpatch).

Generation of Mouse Anti-Rat LV Anti-Serum.

Murine sera generation was conducted following UC Davis institutionalprocedures and was covered by IACUC #15064. Isolated adult Fischer ratLV's (n=6) were homogenized and injected subcutaneously into C57BL/6mice (n=6) on days 0, 14, 28, 42, 56 and 72. Serum was collected at days0, 14, 28, 42, 56, 72, and 84, and stored at −80° C.

Antigen Removal (AR) Procedure.

All steps of the AR protocol are performed at 125 rpm and at RT or 4° C.unless otherwise stated. AR procedure for Water-Soluble antigens (WSA)and Lipid-Soluble antigens (LSA) involves incubating MPs in any given ARadditive such as the examples shown in (Table 1) for 1-3 days, dependingon the additive used. Anatomically adjacent MP pieces are subjected to 1min incubations and serve as negative AR controls. Following nucleicacid digestion for 24 h and washout for 48 h, MPs following AR(designated as MP-AR) are stored in 15% DMSO in DMEM at −80° C.

TABLE 1 ANTIGEN REMOVAL ADDITIVES FOR EACH PROTEIN CLASS AND LEVELS USEDAR Step Additive [Function/Levels] Water-Soluble BARB (Basic AR Buffer);Hypotonic solution containing proteins 0.5 mM Pefabloc (Roche) andprotease inhibitors and 1% AAS in 10 mM Tris-HCl, antibiotic/antimycoticagent; pH 8.0 Cell lysis Water-Soluble optSARB (optimized BARB correctedfor proteins Solubilizing Antigen Removal isotonic/physiologic saltBuffer; 100 mM DTT, 2 mM concentration; protein/antigen MgCl₂, 100 mMKCl in solubilization with use of reducing BARB) reagent (DTT) ActinCytochalasin D in organic 0, 100 nM, 1 μM, 10 μM Depolymerizationsolvent (such as DMSO) or high glucose DMEM Actin Latrunculin B in highglucose 0, 50 nM, 100 nM, 200 nM Depolymerization DMEM Actin MycalolideB in organic 0, 100 nM, 1 μM, 10 μM Depolymerization solvent (such asDMSO) or high glucose DMEM Actin Swinholide A in organic 0, 100 nM, 1μM, 10 μM Depolymerization solvent (such as DMSO) Cardiac Myosin KCl inaqueous buffer 400 mM, 500 mM, 600 mM, 800 mM solubilization containingprotease inhibitors and antibiotics Cardiac Titin KI in aqueous buffer400 mM, 600 mM, 800 mM, 1M solubilization containing protease inhibitorsand antibiotics Lipid-Soluble Amidosulfobetaine 14 Sulfobetainedetergent. 0, 1%, 2%, proteins (ASB-14) 3%, 4%, 5% Lipid-Soluble ASB-16Sulfobetaine detergent. 0, 0.05%, proteins 0.1%, 1%, 2% Lipid-SolubleSulfobetaine 3-12 (SB 3-12) Sulfobetaine Detergent. 0, 0.1%, proteins0.2%, 0.5%, 2% Lipid-Soluble SB 3-14 Sulfobetaine Detergent. 0, 0.1%,proteins 1%, 2%, 4%, 5% [Decellularization/ 1% SDS (w/v) Strong anionicdetergent, literature Literature control] control

Example of Water-Soluble and Lipid-Soluble Antigen Removal on aMyocardial Patch.

All steps are performed at 125 rpm at RT unless otherwise stated.Following MP isolation and recording of wet weights as described above,MPs are incubated in relaxing solution for 2×15 min at 125 rpm, at 4° C.MPs are then incubated in the lipid-soluble antigen removal solutioncomprising of 2% ASB in optimized Standard Antigen Removal Buffer(optSARB; 0.5 mM Pefabloc and 1% AAS in 10 mM Tris-HCl, pH 8.0, 100 mMDTT, 2 mM MgCl₂, 100 mM KCl) for 2 days at 4° C., with one addition offresh solution after 24 hrs. MPs are then washed 2×15 min in optSARB,before proceeding with sarcomere disassembly using actindepolymerization followed by solubilization of sarcomeric components andassociated water-soluble antigens. Specifically, MPs are incubated for 2h at 37° C. in 50 nM Latrunculin B in high-glucose DMEM, washed 2×15 minin optSARB, incubated for 2 h in 0.6 M KCl containing 0.5 mM Pefablocand 1% AAS, washed for 2×15 min in optSARB, incubated for 2 h in 1.0 MKI containing 0.5 mM Pefabloc and 1% AAS, after which they are left inoptSARB at 4° C. overnight.

The following day KCl and KI incubations are repeated as describedabove, with 2×15 min optSARB washes in between them, followed by optSARBwash overnight at 4° C. The next day fresh optSARB is applied to theMP-AR scaffolds for 24 h at 4° C. Following this, MP-AR scaffolds areincubated for 24 h at 4° C. with Nuclease solution (2.5 Kunitz units/mLDNAse I, 7.5 Kunitz units/mL RNAse A, 1% AAS, 0.15 M NaCl, 5 mMMgCl₂-6H₂O in 10 mM Tris-HCl, pH 7.6). Then, they are washed for 48 h inwashing solution (0.5 mM Pefabloc and 1% AAS in 1×TBS) which is changedto fresh solution 1/day and lastly, the scaffolds are stored at 80° C.in 15% v/v DMSO in high glucose DMEM storage solution.

Assessment of Residual Antigenicity in MP-AR Samples.

MP-AR samples and controls are minced and residual proteins extracted.Extracted proteins are subjected to one-dimensional electrophoresis(1-DE) and Western blot (WB) probed with mouse anti-rat native LVpoly-polyclonal anti-serum diluted 1:100 and assessed for IgG positivitywith 1:10,000 hrp-conjugated anti-mouse light chain specific secondaryantibody (Jackson Immunoresearch). Western blots are imaged using FluorChem Xplor CCD bioimaging system and AlphaView image acquisition andanalysis software (Alpha Innotech Corp). Densitometry is used toquantify banding pattern intensity. Residual antigenicity of MP-AR isdefined as the ratio of banding intensity for extracts following 2 d ofAR compared the 1 min AR controls. Overall residual antigenicity foreach AR protocol is calculated with respect to the level of antigenspresent in negative control (examples shown in Table 1).

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. A method of removing immunogenic antigens from atissue, comprising: a) solubilizing water-soluble antigens in the tissueby contacting the tissue with a first solution comprising a firstbuffering agent, a first reducing agent, a first protease inhibitor, andone or more salts suitable for maintaining protein solubility, whereinthe first solution is substantially free of an amphiphile, such that thewater-soluble antigens are dissolved in the first solution; b)separating the tissue from the solubilized water-soluble antigens in thefirst solution; c) solubilizing lipid-soluble antigens in the tissue bycontacting the tissue with a second solution comprising a secondbuffering agent, a second reducing agent, a second protease inhibitor,one or more salts suitable for maintaining protein solubility and anamphiphile, such that the lipid-soluble antigens are dissolved in thesecond solution; and d) separating the tissue from the solubilizedlipid-soluble antigens in the second solution; thereby removingimmunogenic antigens from the tissue.
 2. The method of claim 1, whereinone or more of the steps are repeated.
 3. The method of claim 1, whereinthe immunogenic antigens are selected from the group consisting ofprotein antigens, lipid antigens and carbohydrate antigens.
 4. Themethod of claim 1, wherein the first buffering agent maintains a pH inthe range of about 4-11.
 5. The method of claim 1, wherein the one ormore salts of the first solution comprise a metal halide salt.
 6. Themethod of claim 1, wherein the first reducing agent is selected from thegroup consisting of Tributylphosphine (TBP), beta mercaptoethanol,2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP),cysteine-HCl, and dithiothreitol (DTT).
 7. The method of claim 1,wherein the water-soluble antigens are solubilized in the first solutionthat further comprises a chelation agent.
 8. The method of claim 1,wherein the water-soluble antigens are solubilized in the first solutioncomprising Tris-HCl, dithiothreitol (DTT), the first protease inhibitor,and KCl.
 9. The method of claim 1, wherein the amphiphile of the secondsolution is a sulfobetaine.
 10. The method of claim 9, wherein thesulfobetaine is selected from the group consisting of3-[N,N-Dimethyl(3-myristoylaminopropyl)ammonio]propanesulfonate(amidosulfobetaine-14; ASB-14); amidosulfobetaine-16 (ASB-16);4-n-Octylbenzoylamido-propyl-dimethylammonio sulfobetaine (ASB-C8Ø);3-(N,N-Dimethyloctylammonio) propanesulfonate inner salt (SB3-8);N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-10),N-Decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-12),N-Tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (SB 3-14);3-(N,N-Dimethylpalmitylammonio) propanesulfonate (SB3-16);3-(N,N-Dimethyloctadecylammonio) propanesulfonate (SB3-18);3-(1-Pyridinio)-1-propanesulfonate (NDSB-201); 3-(Benzyldimethylammonio)propanesulfonate (NDSB-256); NDSB-211, NDSB-195, NDSB-221;3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS),3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO), and mixtures thereof.
 11. The method of claim 1, wherein thelipid-soluble antigens are solubilized in the second solution comprisingTris-HCl, dithiothreitol (DTT), the second protease inhibitor, KCl andASB-14.
 12. The method of claim 1, wherein the method yields asubstantially intact extracellular matrix (ECM) compatible with viablecell repopulation.
 13. The method of claim 12, further comprisingrepopulating the ECM with live cells.
 14. The method of claim 1, whereinthe tissue is epithelial tissue, endothelial tissue, muscle tissue, orconnective tissue.
 15. The method of claim 1, wherein the tissue isselected from the group consisting of cardiac muscle tissue, striated orskeletal muscle tissue, or smooth muscle tissue, heart, pericardium,heart valve, vessel, vascular conduit, artery, vein, skin, dermis,pericardium, dura, intestinal submucosa, ligament, tendon, bone,cartilage, ureter, urinary bladder, kidney, skin, lung, liver, andumbilical cord.
 16. The method of claim 1, wherein the tissue is anintact tissue.
 17. The method of claim 1, wherein the tissue is withinor a part of an intact organ.
 18. The method of claim 1, wherein thetissue is intact tissue, the method comprising: a) solubilizingwater-soluble antigens in the tissue by contacting the tissue with thefirst solution comprising Tris-HCl, dithiothreitol (DTT), the firstprotease inhibitor, and KCl, wherein the first solution does notcomprise an amphiphile, such that the water-soluble antigens aredissolved in the first solution; b) separating the tissue from thesolubilized water-soluble antigens in the first solution; c)solubilizing lipid-soluble antigens in the tissue by contacting thetissue with a second solution comprising Tris-HCl, dithiothreitol (DTT),the second protease inhibitor, KCl and ASB 14, such that thelipid-soluble antigens are dissolved in the second solution; and d)separating the tissue from the solubilized lipid-soluble antigens in thesecond solution; thereby removing immunogenic antigens from the intacttissue.
 19. A method of removing immunogenic antigens from a tissue,comprising: a) contacting the tissue with one or more cytoskeletaldestabilizing agents; b) solubilizing water-soluble antigens in thetissue; c) separating the tissue from the solubilized water-solubleantigens; d) solubilizing lipid-soluble antigens in the tissue; and e)separating the tissue from the solubilized lipid-soluble antigens;thereby removing immunogenic antigens from the tissue.
 20. The method ofclaim 19, wherein the one or more cytoskeletal destabilizing agentscomprise one or more actin depolymerization agents.
 21. The method ofclaim 19, wherein the one or more cytoskeletal destabilizing agentscomprise one or more microtubule depolymerization or destabilizingagents.
 22. A method of removing immunogenic antigens from a muscletissue, comprising: a) relaxing the muscle tissue in a relaxing solutioncomprising an energy source molecule; b) contacting the muscle tissuewith one or more cytoskeletal destabilizing agents; c) contacting themuscle tissue with a concentrated salt solution; d) solubilizingwater-soluble antigens in the tissue; e) separating the muscle tissuefrom the solubilized water-soluble antigens; f) solubilizinglipid-soluble antigens in the tissue; and g) separating the muscletissue from the solubilized lipid-soluble antigens; thereby removingimmunogenic antigens from the muscle tissue.
 23. The method of claim 22,wherein one or more of the steps are repeated.
 24. The method of claim22, wherein the steps are performed in an order selected from the groupconsisting of: i) a), b), c), d), e), f) and g); ii) a), f), g), b), c),d) and e); iii) a), d), e), b), c), f) and g); iv) a), d), e), f), g),b) and c); v) a), b), c), d), e), f), g), b), c), and e); and vi) a),f), g), b), c), d), e), f), and g).
 25. The method of claim 22, whereinthe muscle tissue is cardiac muscle tissue, striated or skeletal muscletissue, or smooth muscle tissue.
 26. The method of claim 22, wherein themethod comprises the step of relaxing the muscle tissue prior tocontacting the tissue with one or more cytoskeletal destabilizingagents.
 27. The method of claim 22, wherein the muscle tissue is relaxedin a relaxing solution comprising an energy source molecule.
 28. Themethod of claim 27, wherein the energy source molecule is selected fromthe group consisting of a nucleotide 5′-triphosphate (NTP), adenosine,inosine, aspartate, glutamate, creatine phosphate, a Kreb's cycleprecursor or intermediate, glucose, and dextrose.
 29. The method ofclaim 27, wherein the energy source molecule is a Pyrophosphate (PPi) ora nucleotide 5′-triphosphate (NTP) selected from the group consisting ofadenosine 5′-triphosphate (ATP), inosine 5′-triphosphate (ITP),guanidine 5′-triphosphate (GTP), cytidine 5′-triphosphate (CTP), anduridine 5′-triphosphate (UTP).
 30. The method of claim 27, wherein theenergy source molecule is a precursor of adenosine 5′-triphosphate(ATP).
 31. The method of claim 22, wherein the relaxing solution furthercomprises a calcium ion chelating agent.
 32. The method of claim 22,wherein the relaxing solution further comprises a permeabilizationagent.
 33. The method of claim 22, wherein concentrated salt solutioncomprises one or more salts in a concentration range from about 0.5 M toabout 3.0 M.
 34. The method of claim 22, wherein the concentrated saltsolution comprises one or more metal halide salts.
 35. The method ofclaim 22, wherein sarcomeric constituents are not detectable in themuscle tissue.